Prof Vincenzo Cerundolo

Research Area: Immunology
Keywords: vaccination strategies, NKT cells, dendritic cells, innate immunity, T cells and clinical trials
Web Links:
R837 induced colocalisation of MyD88 with human Toll-like receptor 7 (TLR7) is reduced in cells treated with furin-like protein convertase inhibitor (PC inhibitor).
THP-1 cells expressing HA-tagged TLR7 (shown in red) and FLAG-tagged MyD88 (shown in green) were PMA differentiated for 24 hr in the presence of DMSO or PC inhibitor and then stimulated for 3 hr with R837. Confocal pictures were taken. Scale bars represent 1 μm.

R837 induced colocalisation of MyD88 with human Toll-like receptor 7 (TLR7) is reduced in cells ...

The principal aim of the Research in my laboratory is to gain a better understanding of the mechanisms that control the cell-cell interplay required for optimal expansion and activation of tumour-specific T cell populations and to apply this knowledge to the development of better treatment strategies in cancer patients. Research in my laboratory is divided into three complementary areas:

  • Analysis of tumour-specific immune responses in melanoma patients and the role of the tumour micro-environment in hampering tumour-specific immune responses;
  • Structural, kinetic and functional analyses of invariant NKT (iNKT) cell activation;
  • Clinical trial vaccine programme in melanoma patients.

1) Analysis of the Tumour-Specific Immune Response

By using HLA class I tetramers we have gained an understanding of the tumour-specific immune response, and have been able to show that in some patients with metastatic melanoma there are expanded populations of tumour-specific cytotoxic T lymphocytes.

We have recently started a research programme focused on the role of the tumour micro-environment in suppressing tumour-specific immune responses. The results of our studies have demonstrated the ability of invariant NKT cells to modulate the phenotype of Il-10 secreting myeloid cells in a large proportion of melanoma patients. These cells suppress proliferation and activity of tumour-specific T cell responses (De Santo et al. 2010). More recently, we have extended these results by demonstrating: i) the ability of tumours to up-regulate a novel tryptophan specific transporter to survive in a low nutrients microenvironment (Silk et al. 2011); ii) secretion of arginase II by a large proportion of AML blasts, which significantly hampers T cell and haematopoietic stem cell proliferation (Mussai et al., Blood 2013); In addition, we have initiated a programme to study how tissue stroma can modulate the expression of tissue homing receptors. It is known that the production of retinoic acid (RA) by dendritic cells (DCs) is critical for driving the development of gut-tropic immune responses; however, the factors that regulate RA synthesis by DCs remain poorly defined. We have recently demonstrated the role of prostaglandin E2 (PGE2) in blocking the expression of the retinal dehydrogenases (RALDH), the enzymes responsible for converting vitamin A into RA, and abrogating their ability to induce CCR9 expression upon T cell priming (Stock et al. 2011).

 

2) Structural, Kinetic and Functional Analyses of iNKT Cell Activation

We and others have recently demonstrated that stimulating iNKT cells in vivo with the specific synthetic ligand alpha-GalCer served to significantly enhance immune responses to protein-based vaccines. We have demonstrated that co-injection of iNKT cell agonists together with antigenic proteins enhances antigen-specific T cell responses. This enhancement is dependent on the involvement of iNKT cells and CD1d molecules and requires CD40 signalling. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble antigen by modulating DC function, as recently reviewed. Our results are consistent with the general concept that there is considerable immunostimulatory power in the integration of iNKT-mediated and TLR-mediated signals to DCs (Salio et al. 2007; McCarthy et al. 2007). Understanding this level of regulation will be important in designing appropriate, and hence effective, vaccines.

More recently, in collaboration with Facundo Batista (Cancer Research Institute), we have started to analyse the cross-talk between iNKT cells and B cells (Barral et al. 2010).

We previously developed two novel protocols for the refolding of denatured CD1 molecules, based either on the use of short mono-alkyl detergent molecules or on oxidative refolding chromatography. Techniques developed in the laboratory have enabled the use of ‘refolded’ CD1 molecules to monitor the frequency and phenotype of NKT cells in health and disease. Together with knowledge of the crystal structures of CD1d and CD1b loaded with different lipid antigens (solved in collaboration with Prof. E. Y. Jones, University of Oxford) it has been possible to study both in vitro and in vivo activation of NKT cells, and their effect on the adaptive immune responses.

 

3) Clinical Trial Programme in Melanoma Patients

My group has been developing a very active clinical trial programme to translate our preclinical vaccination strategies into phase I/II clinical trials and several cancer vaccines are currently being compared in the clinic.

Name Department Institution Country
Prof Yvonne Jones STRUBI, Nuffield Department of Clinical Medicine University of Oxford United Kingdom
Frances Platt Deaprtment of Pharmacology University of Oxford United Kingdom
Gurdyal (Del) Besra School of Biosciences University of Birmingham United Kingdom
Prof Mark Middleton Department of Oncology University of Oxford United Kingdom
Dr Adrian L Harris University of Oxford United Kingdom
Cole SL, Dunning J, Kok WL, Benam KH, Benlahrech A, Repapi E, Martinez FO, Drumright L, Powell TJ, Bennett M et al. 2017. M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza. JCI Insight, 2 (7), pp. e91868. | Show Abstract | Read more

In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.

Timosenko E, Hadjinicolaou AV, Cerundolo V. 2017. Modulation of cancer-specific immune responses by amino acid degrading enzymes. Immunotherapy, 9 (1), pp. 83-97. | Show Abstract | Read more

To evade immune destruction, tumors exploit a wide range of immune escape mechanisms, including the induction of an immunosuppressive tumor microenvironment. This is mediated, in part, by amino acid degrading enzymes indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, arginase 1 and arginase 2, which have emerged as key players in the regulation of tumor-induced immune tolerance. Here we describe how the expression of tryptophan- and arginine-degrading enzymes by tumor and tumor-infiltrating cells can hamper cancer-specific immune responses, and discuss how this knowledge is being exploited to develop new strategies for cancer immunotherapy.

Cheung KL, Jarrett R, Subramaniam S, Salimi M, Gutowska-Owsiak D, Chen YL, Hardman C, Xue L, Cerundolo V, Ogg G. 2016. Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a. J Exp Med, 213 (11), pp. 2399-2412. | Show Abstract | Read more

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Jukes JP, Gileadi U, Ghadbane H, Yu TF, Shepherd D, Cox LR, Besra GS, Cerundolo V. 2016. Non-glycosidic compounds can stimulate both human and mouse iNKT cells. Eur J Immunol, 46 (5), pp. 1224-1234. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non-glycosidic CD1d-binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6- or 7-membered ring results in significantly more potent non-glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti-tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α-galactosylceramide (α-GalCer), achieving an enhanced T-cell response at lower concentrations compared with α-GalCer both in vitro, using human iNKT-cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non-glycosidic ThrCer-based analogs that have improved potency in iNKT-cell activation compared with that of α-GalCer, and are clinically relevant iNKT-cell agonists.

Kamaladasa A, Wickramasinghe N, Adikari TN, Gomes L, Shyamali NL, Salio M, Cerundolo V, Ogg GS, Malavige GN. 2016. Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection. Clin Exp Immunol, 185 (2), pp. 228-238. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)-γ and interleukin (IL)-4 ex-vivo enzyme-linked immunospot (ELISPOT) assays following stimulation with alpha-galactosyl-ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4(+) subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus-specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl-6 (P = 0·0003) and both Bcl-6 and inducible T cell co-stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4(+) iNKT cells, with reduced expression of CD161 markers.

Jarrett R, Salio M, Lloyd-Lavery A, Subramaniam S, Bourgeois E, Archer C, Cheung KL, Hardman C, Chandler D, Salimi M et al. 2016. Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase. Sci Transl Med, 8 (325), pp. 325ra18. | Show Abstract | Read more

Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation contribute to pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and although Langerhans cells expressing the nonclassical major histocompatibility complex (MHC) family member CD1a are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. We observed that house dust mite (HDM) allergen generates neolipid antigens presented by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth in individuals with atopic dermatitis and showed rapid effector function, consistent with antigen-driven maturation. In HDM-challenged human skin, we observed phospholipase A2 (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2, and such cells infiltrated the skin after allergen challenge. Moreover, we observed that the skin barrier protein filaggrin, insufficiency of which is associated with atopic skin disease, inhibited PLA2 activity and decreased CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity suggest that nonpeptide stimulants of T cells act as haptens that modify peptides or proteins; however, our results show that HDM proteins may also generate neolipid antigens that directly activate T cells. These data define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach.

Van Rhijn I, van Berlo T, Hilmenyuk T, Cheng TY, Wolf BJ, Tatituri RV, Uldrich AP, Napolitani G, Cerundolo V, Altman JD et al. 2016. Human autoreactive T cells recognize CD1b and phospholipids. Proc Natl Acad Sci U S A, 113 (2), pp. 380-385. | Show Abstract | Read more

In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids.

Nakaya HI, Clutterbuck E, Kazmin D, Wang L, Cortese M, Bosinger SE, Patel NB, Zak DE, Aderem A, Dong T et al. 2016. Systems biology of immunity to MF59-adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood. Proc Natl Acad Sci U S A, 113 (7), pp. 1853-1858. | Show Abstract | Read more

The dynamics and molecular mechanisms underlying vaccine immunity in early childhood remain poorly understood. Here we applied systems approaches to investigate the innate and adaptive responses to trivalent inactivated influenza vaccine (TIV) and MF59-adjuvanted TIV (ATIV) in 90 14- to 24-mo-old healthy children. MF59 enhanced the magnitude and kinetics of serum antibody titers following vaccination, and induced a greater frequency of vaccine specific, multicytokine-producing CD4(+) T cells. Compared with transcriptional responses to TIV vaccination previously reported in adults, responses to TIV in infants were markedly attenuated, limited to genes regulating antiviral and antigen presentation pathways, and observed only in a subset of vaccinees. In contrast, transcriptional responses to ATIV boost were more homogenous and robust. Interestingly, a day 1 gene signature characteristic of the innate response (antiviral IFN genes, dendritic cell, and monocyte responses) correlated with hemagglutination at day 28. These findings demonstrate that MF59 enhances the magnitude, kinetics, and consistency of the innate and adaptive response to vaccination with the seasonal influenza vaccine during early childhood, and identify potential molecular correlates of antibody responses.

Galson JD, Trück J, Clutterbuck EA, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2016. Erratum to: B-cell repertoire dynamics after sequential hepatitis B vaccination and evidence for cross-reactive B-cell activation. Genome Med, 8 (1), pp. 81. | Show Abstract | Read more

© 2016 The Author(s).It has come to our attention that there was an omission in the Acknowledgements section in this article [1]. The Acknowledgements section should read: The authors are grateful to the study participants, to the doctors and nurses at the Oxford Vaccine Group for assisting with sample collection, and to the National Institute for Health Research Clinical Research Network. The authors thank Craig Waugh for help with cell sorting and the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (subsidized by Wellcome Trust grant reference 090532/Z/09/Z) for the generation of sequencing data. Purified HBsAg was provided by GlaxoSmithKline Biologicals SA, and conjugated to APC by Miltenyi Biotec.

Galson JD, Trück J, Clutterbuck EA, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2016. B-cell repertoire dynamics after sequential hepatitis B vaccination and evidence for cross-reactive B-cell activation. Genome Med, 8 (1), pp. 68. | Show Abstract | Read more

BACKGROUND: A diverse B-cell repertoire is essential for recognition and response to infectious and vaccine antigens. High-throughput sequencing of B-cell receptor (BCR) genes can now be used to study the B-cell repertoire at great depth and may shed more light on B-cell responses than conventional immunological methods. Here, we use high-throughput BCR sequencing to provide novel insight into B-cell dynamics following a primary course of hepatitis B vaccination. METHODS: Nine vaccine-naïve participants were administered three doses of hepatitis B vaccine (months 0, 1, and 2 or 7). High-throughput Illumina sequencing of the total BCR repertoire was combined with targeted sequencing of sorted vaccine antigen-enriched B cells to analyze the longitudinal response of both the total and vaccine-specific repertoire after each vaccine. ELISpot was used to determine vaccine-specific cell numbers following each vaccine. RESULTS: Deconvoluting the vaccine-specific from total BCR repertoire showed that vaccine-specific sequence clusters comprised <0.1 % of total sequence clusters, and had certain stereotypic features. The vaccine-specific BCR sequence clusters were expanded after each of the three vaccine doses, despite no vaccine-specific B cells being detected by ELISpot after the first vaccine dose. These vaccine-specific BCR clusters detected after the first vaccine dose had distinct properties compared to those detected after subsequent doses; they were more mutated, present at low frequency even prior to vaccination, and appeared to be derived from more mature B cells. CONCLUSIONS: These results demonstrate the high-sensitivity of our vaccine-specific BCR analysis approach and suggest an alternative view of the B-cell response to novel antigens. In the response to the first vaccine dose, many vaccine-specific BCR clusters appeared to largely derive from previously activated cross-reactive B cells that have low affinity for the vaccine antigen, and subsequent doses were required to yield higher affinity B cells.

Dölen Y, Kreutz M, Gileadi U, Tel J, Vasaturo A, van Dinther EA, van Hout-Kuijer MA, Cerundolo V, Figdor CG. 2016. Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses. Oncoimmunology, 5 (1), pp. e1068493. | Show Abstract | Read more

Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here, we compared the efficacy of the invariant NKT (iNKT) cell agonist α-galactosylceramide (α-GalCer) and TLR ligands (R848 and poly I:C) as an adjuvant for the full length ovalbumin (OVA) in PLGA nanoparticles. We observed that OVA+α-GalCer nanoparticles (NP) are superior over OVA+TLR-L NP in generating and stimulating antigen-specific cytotoxic T lymphocytes without the need for CD4(+) T cell help. Not only a 4-fold higher induction of antigen-specific T cells was observed, but also a more profound IFN-γ secretion was obtained by the addition α-GalCer. Surprisingly, we observed that mixtures of OVA containing NP with α-GalCer were ineffective, demonstrating that co-encapsulation of both α-GalCer and antigen within the same nanoparticle is essential for the observed T cell responses. Moreover, a single immunization with OVA+α-GalCer NP provided substantial protection from tumor formation and even delayed the growth of already established tumors, which coincided with a prominent and enhanced antigen-specific CD8(+) T cell infiltration. The provided evidence on the advantage of antigen and α-GalCer coencapsulation should be considered in the design of future nanoparticle vaccines for therapeutic purposes.

Galson JD, Trück J, Fowler A, Clutterbuck EA, Münz M, Cerundolo V, Reinhard C, van der Most R, Pollard AJ, Lunter G, Kelly DF. 2015. Analysis of B Cell Repertoire Dynamics Following Hepatitis B Vaccination in Humans, and Enrichment of Vaccine-specific Antibody Sequences. EBioMedicine, 2 (12), pp. 2070-2079. | Show Abstract | Read more

Generating a diverse B cell immunoglobulin repertoire is essential for protection against infection. The repertoire in humans can now be comprehensively measured by high-throughput sequencing. Using hepatitis B vaccination as a model, we determined how the total immunoglobulin sequence repertoire changes following antigen exposure in humans, and compared this to sequences from vaccine-specific sorted cells. Clonal sequence expansions were seen 7 days after vaccination, which correlated with vaccine-specific plasma cell numbers. These expansions caused an increase in mutation, and a decrease in diversity and complementarity-determining region 3 sequence length in the repertoire. We also saw an increase in sequence convergence between participants 14 and 21 days after vaccination, coinciding with an increase of vaccine-specific memory cells. These features allowed development of a model for in silico enrichment of vaccine-specific sequences from the total repertoire. Identifying antigen-specific sequences from total repertoire data could aid our understanding B cell driven immunity, and be used for disease diagnostics and vaccine evaluation.

Seder R, Reed SG, O'Hagan D, Malyala P, D'Oro U, Laera D, Abrignani S, Cerundolo V, Steinman L, Bertholet S. 2015. Gaps in knowledge and prospects for research of adjuvanted vaccines. Vaccine, 33 Suppl 2 (S2), pp. B40-B43. | Show Abstract | Read more

A panel of researchers working in different areas of adjuvanted vaccines deliberated over the topic, "Gaps in knowledge and prospects for research of adjuvanted vaccines" at, "Enhancing Vaccine Immunity and Value" conference held in July 2014. Several vaccine challenges and applications for new adjuvant technologies were discussed.

Galson JD, Clutterbuck EA, Trück J, Ramasamy MN, Münz M, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2015. BCR repertoire sequencing: different patterns of B-cell activation after two Meningococcal vaccines. Immunol Cell Biol, 93 (10), pp. 885-895. | Show Abstract | Read more

Next-generation sequencing was used to investigate the B-cell receptor heavy chain transcript repertoire of different B-cell subsets (naive, marginal zone (MZ), immunoglobulin M (IgM) memory and IgG memory) at baseline, and of plasma cells (PCs) 7 days following administration of serogroup ACWY meningococcal polysaccharide and protein-polysaccharide conjugate vaccines. Baseline B-cell subsets could be distinguished from each other using a small number of repertoire properties (clonality, mutation from germline and complementarity-determining region 3 (CDR3) length) that were conserved between individuals. However, analyzing the CDR3 amino-acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 PCs demonstrated nearly 10-fold greater sequence sharing between individuals than the baseline subsets, consistent with the PCs being induced by the vaccine antigen and sharing specificity for a more limited range of epitopes. By annotating PC sequences based on IgG subclass usage and mutation, and also comparing them with the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. PCs produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the PCs were predominantly IgG2, less mutated and were equally likely to be related to MZ, IgM memory or IgG memory cells. High-throughput B-cell repertoire sequencing thus provides a unique insight into patterns of B-cell activation not possible from more conventional measures of immunogenicity.

McEwen-Smith RM, Salio M, Cerundolo V. 2015. The regulatory role of invariant NKT cells in tumor immunity. Cancer Immunol Res, 3 (5), pp. 425-435. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are a unique population of T lymphocytes, which lie at the interface between the innate and adaptive immune systems, and are important mediators of immune responses and tumor surveillance. iNKT cells recognize lipid antigens in a CD1d-dependent manner; their subsequent activation results in a rapid and specific downstream response, which enhances both innate and adaptive immunity. The capacity of iNKT cells to modify the immune microenvironment influences the ability of the host to control tumor growth, making them an important population to be harnessed in the clinic for the development of anticancer therapeutics. Indeed, the identification of strong iNKT-cell agonists, such as α-galactosylceramide (α-GalCer) and its analogues, has led to the development of synthetic lipids that have shown potential in vaccination and treatment against cancers. In this Masters of Immunology article, we discuss these latest findings and summarize the major discoveries in iNKT-cell biology, which have enabled the design of potent strategies for immune-mediated tumor destruction.

Hipp MM, Shepherd D, Booth S, Waithe D, Reis e Sousa C, Cerundolo V. 2015. The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes. J Immunol, 194 (11), pp. 5417-5425. | Show Abstract | Read more

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA.

McEwen-Smith RM, Salio M, Cerundolo V. 2015. CD1d-dependent endogenous and exogenous lipid antigen presentation. Curr Opin Immunol, 34 pp. 116-125. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipids in the context of CD1d molecules, and through the activation and maturation of dendritic cells and B cells, can significantly enhance priming of antigen-specific T and B cell responses. Recent findings have provided important insights into the recognition of several novel endogenous lipids by iNKT cells, and into the mechanisms controlling their generation and loading onto CD1d molecules. In this review we discuss these latest findings and describe the role of autophagy in iNKT cell development and activation.

Brodovitch A, Shenderov E, Cerundolo V, Bongrand P, Pierres A, van der Merwe PA. 2015. T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR-binding parameters. Eur J Immunol, 45 (6), pp. 1635-1642. | Show Abstract | Read more

T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide-MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

Stranks AJ, Hansen AL, Panse I, Mortensen M, Ferguson DJ, Puleston DJ, Shenderov K, Watson AS, Veldhoen M, Phadwal K et al. 2015. Autophagy Controls Acquisition of Aging Features in Macrophages. J Innate Immun, 7 (4), pp. 375-391. | Show Abstract | Read more

Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as 'inflamm-aging'. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased - a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging.

Salio M, Cerundolo V. 2015. NU-dependent B-cell activation in Gaucher disease BLOOD, 125 (8), pp. 1200-1202.

Salio M, Cerundolo V. 2015. NKT-dependent B-cell activation in Gaucher disease. Blood, 125 (8), pp. 1200-1202. | Show Abstract | Read more

In this issue of Blood, Nair et al describe a new population of type II natural killer T (NKT) cells with follicular helper phenotype (TFH), which is more abundant in patients and mice with Gaucher disease (GD) and is capable of regulating B-cell activity.

Bourgeois EA, Subramaniam S, Cheng TY, De Jong A, Layre E, Ly D, Salimi M, Legaspi A, Modlin RL, Salio M et al. 2015. Bee venom processes human skin lipids for presentation by CD1a. J Exp Med, 212 (2), pp. 149-163. | Show Abstract | Read more

Venoms frequently co-opt host immune responses, so study of their mode of action can provide insight into novel inflammatory pathways. Using bee and wasp venom responses as a model system, we investigated whether venoms contain CD1-presented antigens. Here, we show that venoms activate human T cells via CD1a proteins. Whereas CD1 proteins typically present lipids, chromatographic separation of venoms unexpectedly showed that stimulatory factors partition into protein-containing fractions. This finding was explained by demonstrating that bee venom-derived phospholipase A2 (PLA2) activates T cells through generation of small neoantigens, such as free fatty acids and lysophospholipids, from common phosphodiacylglycerides. Patient studies showed that injected PLA2 generates lysophospholipids within human skin in vivo, and polyclonal T cell responses are dependent on CD1a protein and PLA2. These findings support a previously unknown skin immune response based on T cell recognition of CD1a proteins and lipid neoantigen generated in vivo by phospholipases. The findings have implications for skin barrier sensing by T cells and mechanisms underlying phospholipase-dependent inflammatory skin disease.

Galson JD, Clutterbuck EA, Trück J, Ramasamy MN, Münz M, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2015. BCR repertoire sequencing: Different patterns of B-cell activation after two Meningococcal vaccines Immunology and Cell Biology, 93 (10), pp. 885-895. | Show Abstract | Read more

© 2015 Australasian Society for Immunology Inc. All rights reserved.Next-generation sequencing was used to investigate the B-cell receptor heavy chain transcript repertoire of different B-cell subsets (naive, marginal zone (MZ), immunoglobulin M (IgM) memory and IgG memory) at baseline, and of plasma cells (PCs) 7 days following administration of serogroup ACWY meningococcal polysaccharide and protein-polysaccharide conjugate vaccines. Baseline B-cell subsets could be distinguished from each other using a small number of repertoire properties (clonality, mutation from germline and complementarity-determining region 3 (CDR3) length) that were conserved between individuals. However, analyzing the CDR3 amino-acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 PCs demonstrated nearly 10-fold greater sequence sharing between individuals than the baseline subsets, consistent with the PCs being induced by the vaccine antigen and sharing specificity for a more limited range of epitopes. By annotating PC sequences based on IgG subclass usage and mutation, and also comparing them with the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. PCs produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the PCs were predominantly IgG2, less mutated and were equally likely to be related to MZ, IgM memory or IgG memory cells. High-throughput B-cell repertoire sequencing thus provides a unique insight into patterns of B-cell activation not possible from more conventional measures of immunogenicity.

Galson JD, Trück J, Fowler A, Münz M, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2015. In-Depth Assessment of Within-Individual and Inter-Individual Variation in the B Cell Receptor Repertoire. Front Immunol, 6 (OCT), pp. 531. | Show Abstract | Read more

High-throughput sequencing of the B cell receptor (BCR) repertoire can provide rapid characterization of the B cell response in a wide variety of applications in health, after vaccination and in infectious, inflammatory and immune-driven disease, and is starting to yield clinical applications. However, the interpretation of repertoire data is compromised by a lack of studies to assess the intra and inter-individual variation in the BCR repertoire over time in healthy individuals. We applied a standardized isotype-specific BCR repertoire deep sequencing protocol to a single highly sampled participant, and then evaluated the method in 9 further participants to comprehensively describe such variation. We assessed total repertoire metrics of mutation, diversity, VJ gene usage and isotype subclass usage as well as tracking specific BCR sequence clusters. There was good assay reproducibility (both in PCR amplification and biological replicates), but we detected striking fluctuations in the repertoire over time that we hypothesize may be due to subclinical immune activation. Repertoire properties were unique for each individual, which could partly be explained by a decrease in IgG2 with age, and genetic differences at the immunoglobulin locus. There was a small repertoire of public clusters (0.5, 0.3, and 1.4% of total IgA, IgG, and IgM clusters, respectively), which was enriched for expanded clusters containing sequences with suspected specificity toward antigens that should have been historically encountered by all participants through prior immunization or infection. We thus provide baseline BCR repertoire information that can be used to inform future study design, and aid in interpretation of results from these studies. Furthermore, our results indicate that BCR repertoire studies could be used to track changes in the public repertoire in and between populations that might relate to population immunity against infectious diseases, and identify the characteristics of inflammatory and immunological diseases.

Stranks AJ, Hansen AL, Panse I, Mortensen M, Ferguson DJP, Puleston DJ, Shenderov K, Watson AS, Veldhoen M, Phadwal K et al. 2015. Autophagy Controls Acquisition of Aging Features in Macrophages Journal of Innate Immunity, 7 (4), pp. 375-391. | Show Abstract | Read more

© 2015 S. Karger AG, Basel.Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as 'inflamm-aging'. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased-a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging.

Salio M, Cerundolo V. 2015. Regulation of Lipid Specific and Vitamin Specific Non-MHC Restricted T Cells by Antigen Presenting Cells and Their Therapeutic Potentials. Front Immunol, 6 (JUL), pp. 388. | Show Abstract | Read more

Since initial reports, more than 25 years ago, that T cells recognize lipids in the context on non-polymorphic CD1 molecules, our understanding of antigen presentation to non-peptide-specific T cell populations has deepened. It is now clear that αβ T cells bearing semi-invariant T cell receptor, as well as subsets of γδ T cells, recognize a variety of self and non-self lipids and contribute to shaping immune responses via cross talk with dendritic cells and B cells. Furthermore, it has been demonstrated that small molecules derived from the microbial riboflavin biosynthetic pathway (vitamin B2) bind monomorphic MR1 molecules and activate mucosal-associated invariant T cells, another population of semi-invariant T cells. Novel insights in the biological relevance of non-peptide-specific T cells have emerged with the development of tetrameric CD1 and MR1 molecules, which has allowed accurate enumeration and functional analysis of CD1- and MR1-restricted T cells in humans and discovery of novel populations of semi-invariant T cells. The phenotype and function of non-peptide-specific T cells will be discussed in the context of the known distribution of CD1 and MR1 molecules by different subsets of antigen-presenting cells at steady state and following infection. Concurrent modulation of CD1 transcription and lipid biosynthetic pathways upon TLR stimulation, coupled with efficient lipid antigen processing, result in the increased cell surface expression of antigenic CD1-lipid complexes. Similarly, MR1 expression is almost undetectable in resting APC and it is upregulated following bacterial infection, likely due to stabilization of MR1 molecules by microbial antigens. The tight regulation of CD1 and MR1 expression at steady state and during infection may represent an important mechanism to limit autoreactivity, while promoting T cell responses to foreign antigens.

Brodovitch A, Shenderov E, Cerundolo V, Bongrand P, Pierres A, van der Merwe PA. 2015. T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR-binding parameters European Journal of Immunology, 45 (6), pp. 1635-1642. | Show Abstract | Read more

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide-MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

Galluzzi L, Vacchelli E, Bravo-San Pedro JM, Buqué A, Senovilla L, Baracco EE, Bloy N, Castoldi F, Abastado JP, Agostinis P et al. 2014. Classification of current anticancer immunotherapies. Oncotarget, 5 (24), pp. 12472-12508. | Show Abstract | Read more

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and "active" based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.

Salio M, Puleston DJ, Mathan TS, Shepherd D, Stranks AJ, Adamopoulou E, Veerapen N, Besra GS, Hollander GA, Simon AK, Cerundolo V. 2014. Essential role for autophagy during invariant NKT cell development. Proc Natl Acad Sci U S A, 111 (52), pp. E5678-E5687. | Show Abstract | Read more

Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7(-/-)), thymic iNKT cell development--unlike conventional T-cell development--is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell-intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8(+) T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion.

Cerundolo V. 2014. Harnessing invariant NKT-cells to enhance antigen specific immune responses IMMUNOLOGY, 143 pp. 16-16.

Galson JD, Clutterbuck EA, Trueck J, Muenz M, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2014. Plasma cell antibody repertoire analysis following administration of meningococcal polysaccharide and protein-polysaccharide conjugate vaccines: evidence of distinct patterns of B cell activation IMMUNOLOGY, 143 pp. 62-62.

Chen JL, Dawoodji A, Tarlton A, Gnjatic S, Tajar A, Karydis I, Browning J, Pratap S, Verfaille C, Venhaus RR et al. 2015. NY-ESO-1 specific antibody and cellular responses in melanoma patients primed with NY-ESO-1 protein in ISCOMATRIX and boosted with recombinant NY-ESO-1 fowlpox virus. Int J Cancer, 136 (6), pp. E590-E601. | Show Abstract | Read more

Vaccination strategies based on repeated injections of NY-ESO-1 protein formulated in ISCOMATRIX particles (NY-ESO-1 ISCOMATRIX) have shown to elicit combined NY-ESO-1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime-boost strategies based on the combination with NY-ESO-1 ISCOMATRIX with different NY-ESO-1 boosting reagents could be used to increase NY-ESO-1 CD8(+) or CD4(+) T cell responses. To address this question, we carried out a randomized clinical trial in 39 high-risk, resected melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY-ESO-1 (rF-NY-ESO-1) (Arm A) or NY-ESO-1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY-ESO-1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY-ESO-1 ISCOMATRIX alone elicited a strong NY-ESO-1 specific CD4(+) T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8(+) T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF-NY-ESO-1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8(+) T cell responses. In addition, our results clearly identified immunodominant regions in the NY-ESO-1 protein: NY-ESO-179-102 and NY-ESO-1115-138 for CD4+ T cells and NY-ESO-185-108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY-ESO-1 protein should be considered in future clinical trials as immunodominant epitopes.

Dawoodji A, Chen JL, Shepherd D, Dalin F, Tarlton A, Alimohammadi M, Penna-Martinez M, Meyer G, Mitchell AL, Gan EH et al. 2014. High frequency of cytolytic 21-hydroxylase-specific CD8+ T cells in autoimmune Addison's disease patients. J Immunol, 193 (5), pp. 2118-2126. | Show Abstract | Read more

The mechanisms behind destruction of the adrenal glands in autoimmune Addison's disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase-specific T cells detectable in 20 patients with Addison's disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8(+) and CD4(+) T cell responses in a large proportion of Addison's patients both ex vivo and after in vitro culture of PBLs ≤20 y after diagnosis. In a large proportion of patients, CD8(+) and CD4(+) 21-hydroxylase-specific T cells were very abundant and detectable in ex vivo assays. HLA class I tetramer-guided isolation of 21-hydroxylase-specific CD8(+) T cells showed their ability to lyse 21-hydroxylase-positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer.

Shenderov K, Riteau N, Yip R, Mayer-Barber KD, Oland S, Hieny S, Fitzgerald P, Oberst A, Dillon CP, Green DR et al. 2014. Cutting edge: Endoplasmic reticulum stress licenses macrophages to produce mature IL-1β in response to TLR4 stimulation through a caspase-8- and TRIF-dependent pathway. J Immunol, 192 (5), pp. 2029-2033. | Show Abstract | Read more

The accumulation of improperly folded proteins within the endoplasmic reticulum (ER) generates perturbations known as ER stress that engage the unfolded protein response. ER stress is involved in many inflammatory pathologies that are also associated with the production of the proinflammatory cytokine IL-1β. In this study, we demonstrate that macrophages undergoing ER stress are able to drive the production and processing of pro-IL-1β in response to LPS stimulation in vitro. Interestingly, the classical NLRP3 inflammasome is dispensable, because maturation of pro-IL-1β occurs normally in the absence of the adaptor protein ASC. In contrast, processing of pro-IL-1β is fully dependent on caspase-8. Intriguingly, we found that neither the unfolded protein response transcription factors XBP1 and CHOP nor the TLR4 adaptor molecule MyD88 is necessary for caspase-8 activation. Instead, both caspase activation and IL-1β production require the alternative TLR4 adaptor TRIF. This pathway may contribute to IL-1-driven tissue pathology in certain disease settings.

Pan X, Huang LC, Dong T, Peng Y, Cerundolo V, McGowan S, Ogg G. 2014. Combinatorial HLA-peptide bead libraries for high throughput identification of CD8+ T cell specificity Journal of Immunological Methods, 403 (1-2), pp. 72-78. | Show Abstract | Read more

Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to CD8-null HLA-A*0201 on beads, we probed HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. We observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells. © 2013 Elsevier B.V.

Salio M, Silk JD, Jones EY, Cerundolo V. 2014. Biology of CD1- and MR1-restricted T cells. Annu Rev Immunol, 32 (1), pp. 323-366. | Show Abstract | Read more

Over the past 15 years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I-like molecules, CD1 proteins. In this review, we describe the most recent events in the field, with particular emphasis on (a) structural and functional aspects of lipid presentation by CD1 molecules, (b) the development of CD1d-restricted invariant natural killer T (iNKT) cells and transcription factors required for their differentiation, (c) the ability of iNKT cells to modulate innate and adaptive immune responses through their cross talk with lymphoid and myeloid cells, and (d) MR1-restricted and group I (CD1a, CD1b, and CD1c)-restricted T cells.

Puleston DJ, Zhang H, Powell TJ, Lipina E, Sims S, Panse I, Watson AS, Cerundolo V, Townsend AR, Klenerman P, Simon AK. 2014. Autophagy is a critical regulator of memory CD8(+) T cell formation. Elife, 3 | Show Abstract | Read more

During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

Pan X, Huang LC, Dong T, Peng Y, Cerundolo V, McGowan S, Ogg G. 2014. Combinatorial HLA-peptide bead libraries for high throughput identification of CD8⁺ T cell specificity. J Immunol Methods, 403 (1-2), pp. 72-78. | Show Abstract | Read more

Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to CD8-null HLA-A*0201 on beads, we probed HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. We observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells.

Salio M, Ghadbane H, Dushek O, Shepherd D, Cypen J, Gileadi U, Aichinger MC, Napolitani G, Qi X, van der Merwe PA et al. 2013. Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation. Proc Natl Acad Sci U S A, 110 (49), pp. E4753-E4761. | Show Abstract | Read more

Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the off-rate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a "lipid editor," capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity.

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Scopus

Scher JU, Sczesnak A, Longman RS, Segata N, Ubeda C, Bielski C, Rostron T, Cerundolo V, Pamer EG, Abramson SB et al. 2013. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis eLife, 2013 (2), | Show Abstract | Read more

Rheumatoid arthritis (RA) is a prevalent systemic autoimmune disease, caused by a combination of genetic and environmental factors. Animal models suggest a role for intestinal bacteria in supporting the systemic immune response required for joint inflammation. Here we performed 16S sequencing on 114 stool samples from rheumatoid arthritis patients and controls, and shotgun sequencing on a subset of 44 such samples. We identified the presence of Prevotella copri as strongly correlated with disease in new-onset untreated rheumatoid arthritis (NORA) patients. Increases in Prevotella abundance correlated with a reduction in Bacteroides and a loss of reportedly beneficial microbes in NORA subjects. We also identified unique Prevotella genes that correlated with disease. Further, colonization of mice revealed the ability of P. copri to dominate the intestinal microbiota and resulted in an increased sensitivity to chemically induced colitis. This work identifies a potential role for P. copri in the pathogenesis of RA. © Scher et al.

Hipp MM, Shepherd D, Gileadi U, Aichinger MC, Kessler BM, Edelmann MJ, Essalmani R, Seidah NG, Reis e Sousa C, Cerundolo V. 2013. Processing of Human Toll-like Receptor 7 by Furin-like Proprotein Convertases Is Required for Its Accumulation and Activity in Endosomes IMMUNITY, 39 (4), pp. 711-721. | Read more

Duman M, Chtcheglova LA, Zhu R, Bozna BL, Polzella P, Cerundolo V, Hinterdorfer P. 2013. Nanomapping of CD1d-glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging. J Mol Recognit, 26 (9), pp. 408-414. | Show Abstract | Read more

CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.

Crawford G, Enders A, Gileadi U, Stankovic S, Zhang Q, Lambe T, Crockford TL, Lockstone HE, Freeman A, Arkwright PD et al. 2013. DOCK8 is critical for the survival and function of NKT cells. Blood, 122 (12), pp. 2052-2061. | Show Abstract | Read more

Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1(+) NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.

Mussai F, De Santo C, Abu-Dayyeh I, Booth S, Quek L, McEwen-Smith RM, Qureshi A, Dazzi F, Vyas P, Cerundolo V. 2013. Acute myeloid leukemia creates an arginase-dependent immunosuppressive microenvironment. Blood, 122 (5), pp. 749-758. | Show Abstract | Read more

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the second most common frequent leukemia of childhood. Patients may present with lymphopenia or pancytopenia at diagnosis. We investigated the mechanisms by which AML causes pancytopenia and suppresses patients' immune response. This study identified for the first time that AML blasts alter the immune microenvironment through enhanced arginine metabolism. Arginase II is expressed and released from AML blasts and is present at high concentrations in the plasma of patients with AML, resulting in suppression of T-cell proliferation. We extended these results by demonstrating an arginase-dependent ability of AML blasts to polarize surrounding monocytes into a suppressive M2-like phenotype in vitro and in engrafted nonobese diabetic-severe combined immunodeficiency mice. In addition, AML blasts can suppress the proliferation and differentiation of murine granulocyte-monocyte progenitors and human CD34(+) progenitors. Finally, the study showed that the immunosuppressive activity of AML blasts can be modulated through small-molecule inhibitors of arginase and inducible nitric oxide synthase, suggesting a novel therapeutic target in AML. The results strongly support the hypothesis that AML creates an immunosuppressive microenvironment that contributes to the pancytopenia observed at diagnosis.

Hipp MM, Shepherd D, Gileadi U, Aichinger MC, Kessler BM, Edelmann MJ, Essalmani R, Seidah NG, Reis e Sousa C, Cerundolo V. 2013. Processing of human toll-like receptor 7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes. Immunity, 39 (4), pp. 711-721. | Show Abstract | Read more

Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.

Armitage AE, Gileadi U, Stacey A, Giannoulatou E, Marshall E, Sturges P, Eddowes L, Cerundolo V, Townsend A, Webster C et al. 2013. HEPCIDIN REGULATION DURING ACUTE INFECTIONS AMERICAN JOURNAL OF HEMATOLOGY, 88 (5), pp. E135-E135.

Jervis PJ, Polzella P, Wojno J, Jukes JP, Ghadbane H, Garcia Diaz YR, Besra GS, Cerundolo V, Cox LR. 2013. Design, synthesis, and functional activity of labeled CD1d glycolipid agonists. Bioconjug Chem, 24 (4), pp. 586-594. | Show Abstract | Read more

Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR-α-GalCer-CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR-glycolipid-CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists.

Stock A, Napolitani G, Cerundolo V. 2013. Intestinal DC in migrational imprinting of immune cells. Immunol Cell Biol, 91 (3), pp. 240-249. | Show Abstract | Read more

Dendritic cells (DCs) have a pivotal role in instructing antigen-specific immune responses, processing and presenting antigens to CD4(+) and CD8(+) T cells and producing factors capable to modulate the quality of T-cell responses. In this review, we will provide an historic overview on the identification of the mechanisms controlling lymphocyte migration into the largest immune organ of the body: the gut, and we will describe how in recent years an unexpected role for DCs has emerged as the architects in programming gut-homing immune cells. Specifically, we will review how intestinal DCs utilize the dietary vitamin A metabolite retinoic acid (RA) to program gut-homing lymphocytes and how intestinal DCs acquire the unique capacity to become RA producers.

Scher JU, Sczesnak A, Longman RS, Segata N, Ubeda C, Bielski C, Rostron T, Cerundolo V, Pamer EG, Abramson SB et al. 2013. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. Elife, 2 pp. e01202. | Show Abstract | Read more

Rheumatoid arthritis (RA) is a prevalent systemic autoimmune disease, caused by a combination of genetic and environmental factors. Animal models suggest a role for intestinal bacteria in supporting the systemic immune response required for joint inflammation. Here we performed 16S sequencing on 114 stool samples from rheumatoid arthritis patients and controls, and shotgun sequencing on a subset of 44 such samples. We identified the presence of Prevotella copri as strongly correlated with disease in new-onset untreated rheumatoid arthritis (NORA) patients. Increases in Prevotella abundance correlated with a reduction in Bacteroides and a loss of reportedly beneficial microbes in NORA subjects. We also identified unique Prevotella genes that correlated with disease. Further, colonization of mice revealed the ability of P. copri to dominate the intestinal microbiota and resulted in an increased sensitivity to chemically induced colitis. This work identifies a potential role for P. copri in the pathogenesis of RA. DOI: http://dx.doi.org/10.7554/eLife.01202.001.

Davis LC, Morgan AJ, Chen JL, Snead CM, Bloor-Young D, Shenderov E, Stanton-Humphreys MN, Conway SJ, Churchill GC, Parrington J et al. 2012. NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing. Curr Biol, 22 (24), pp. 2331-2337. | Show Abstract | Read more

A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca(2+)-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the endoplasmic reticulum activates the store-operated Ca(2+)-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus. Here we identify the Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs), as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca(2+) stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca(2+) signals induced by IP(3) or ionomycin, suggesting that critical, local Ca(2+) nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca(2+) signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts.

Porubsky S, Speak AO, Salio M, Jennemann R, Bonrouhi M, Zafarulla R, Singh Y, Dyson J, Luckow B, Lehuen A et al. 2012. Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells. J Immunol, 189 (6), pp. 3007-3017. | Show Abstract | Read more

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.

Mussai F, De Santo C, Cerundolo V. 2012. Interaction between invariant NKT cells and myeloid-derived suppressor cells in cancer patients: evidence and therapeutic opportunities. J Immunother, 35 (6), pp. 449-459. | Show Abstract | Read more

Despite advances in therapeutic strategies, the ability of cancer cells to evade destruction remains a significant obstacle to the development of effective anticancer treatment. In recent years a subset of immune cells, myeloid-derived suppressor cells (MDSCs), has been shown to play a key role in evasion of the patient's immune system by tumor cells. A number of different tumor types are associated with increased numbers of circulating MDSCs in cancer patients, suppressing the immune response and permitting continued tumor cell proliferation. Invariant NKT (iNKT) cells have recently been defined as a unique subset of immune cells that are able to act as a link between the innate and adaptive arms of the immune system. iNKT cells have the ability to carry out immune surveillance of tumor cells and control proliferation of malignant cells. Recently, we presented evidence that iNKT cells are able to interact with and decrease the numbers of circulating MDSCs in melanoma patients. This review discusses the evidence for MDSCs in tumor progression and the implication that iNKT cells could be developed as a potent therapeutic strategy.

Speak AO, Platt N, Salio M, te Vruchte D, Smith DA, Shepherd D, Veerapen N, Besra GS, Yanjanin NM, Simmons L et al. 2012. Invariant natural killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C. Eur J Immunol, 42 (7), pp. 1886-1892. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development.

Barral P, Sánchez-Niño MD, van Rooijen N, Cerundolo V, Batista FD. 2012. The location of splenic NKT cells favours their rapid activation by blood-borne antigen. EMBO J, 31 (10), pp. 2378-2390. | Show Abstract | Read more

Natural killer T (NKT) cells play an important role in mounting protective responses to blood-borne infections. However, though the spleen is the largest blood filter in the body, the distribution and dynamics of NKT cells within this organ are not well characterized. Here we show that the majority of NKT cells patrol around the marginal zone (MZ) and red pulp (RP) of the spleen. In response to lipid antigen, these NKT cells become arrested and rapidly produce cytokines, while the small proportion of NKT cells located in the white pulp (WP) exhibit limited activation. Importantly, disruption of the splenic MZ by chemical or genetic approaches results in a severe reduction in NKT cell activation indicating the need of cooperation between both MZ macrophages and dendritic cells for efficient NKT cell responses. Thus, the location of splenic NKT cells in the MZ and RP facilitates their access to blood-borne antigen and enables the rapid initiation of protective immune responses.

Spiliotis I, Kelley T, Barker C, Achaibar K, Robertson K, Cindrova-Davies T, Burton G, Charnock-Jones S, Ibitoye RT, Ibitoye RT et al. 2012. Proceedings of research in clinical practice 2011. QJM, 105 (6), pp. 593-606. | Show Abstract | Read more

The skeletal muscle proteins, dysferlin (DYSF) and myoferlin (MYOF) have been demonstrated by mass spectrometry to be present in the apical syncytiotrophoblast of the human placenta, where they may play a role in membrane repair. Immunohistochemistry and immunoblotting were performed on placental samples to localize and quantify DYSF and MYOF in first trimester, term and pathological pregnancies. Here, we show that placental DYSF and MYOF are reduced in labour but only MYOF is elevated in cases of intrauterine growth restriction (IUGR) and pre-eclampsia, relative to Caesarean section controls. In term villous explants cultured for 24 h, DYSF levels decreased, compared to T(0) controls, while MYOF levels remained static. Additionally, trophoblastic BeWo cells show reduced levels of DYSF but unchanged levels of MYOF under conditions of oxidative stress, while DYSF levels in microparticles isolated from the supernatant increase. These findings suggest that DYSF but not MYOF plays a pivotal role in membrane repair mechanisms in the human placenta and that dysferlin microparticles could potentially represent a novel biomarker for pre-eclampsia. Ethical approval: Samples were collected with informed written consent of the patients and Local Research Committee approval. Conflict of interest: none declared.

Jervis PJ, Moulis M, Jukes JP, Ghadbane H, Cox LR, Cerundolo V, Besra GS. 2012. Towards multivalent CD1d ligands: synthesis and biological activity of homodimeric α-galactosyl ceramide analogues. Carbohydr Res, 356 pp. 152-162. | Show Abstract | Read more

A library of dimeric CD1d ligands, containing two α-galactosyl ceramide (α-GalCer) units linked by spacers of varying lengths has been synthesised. The key dimerisation reactions were carried out via copper-catalysed click reactions between a 6"-azido-6"-deoxy-α-galactosyl ceramide derivative and various diynes. Each α-GalCer dimer was tested for its ability to stimulate iNKT cells.

Wojno J, Jukes JP, Ghadbane H, Shepherd D, Besra GS, Cerundolo V, Cox LR. 2012. Amide analogues of CD1d agonists modulate iNKT-cell-mediated cytokine production. ACS Chem Biol, 7 (5), pp. 847-855. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical iNKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of iNKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor-α-GalCer-CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon iNKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human iNKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue.

Robert P, Aleksic M, Dushek O, Cerundolo V, Bongrand P, van der Merwe PA. 2012. Kinetics and mechanics of two-dimensional interactions between T cell receptors and different activating ligands. Biophys J, 102 (2), pp. 248-257. | Show Abstract | Read more

Adaptive immune responses are driven by interactions between T cell antigen receptors (TCRs) and complexes of peptide antigens (p) bound to Major Histocompatibility Complex proteins (MHC) on the surface of antigen-presenting cells. Many experiments support the hypothesis that T cell response is quantitatively and qualitatively dependent on the so-called strength of TCR/pMHC association. Most available data are correlations between binding parameters measured in solution (three-dimensional) and pMHC activation potency, suggesting that full lymphocyte activation required a minimal lifetime for TCR/pMHC interaction. However, recent reports suggest important discrepancies between the binding properties of ligand-receptor couples measured in solution (three-dimensional) and those measured using surface-bound molecules (two-dimensional). Other reports suggest that bond mechanical strength may be important in addition to kinetic parameters. Here, we used a laminar flow chamber to monitor at the single molecule level the two-dimensional interaction between a recombinant human TCR and eight pMHCs with variable potency. We found that 1), two-dimensional dissociation rates were comparable to three-dimensional parameters previously obtained with the same molecules; 2), no significant correlation was found between association rates and activating potency of pMHCs; 3), bond mechanical strength was partly independent of bond lifetime; and 4), a suitable combination of bond lifetime and bond strength displayed optimal correlation with activation efficiency. These results suggest possible refinements of contemporary models of signal generation by T cell receptors. In conclusion, we reported, for the first time to our knowledge, the two-dimensional binding properties of eight TCR/pMHC couples in a cell-free system with single bond resolution.

Srivastava PK, Cerundolo V, Knuth A, Allison JP. 2012. An open invitation to the cancer immunology community. Cancer Immun, 12 pp. 1.

Silk KM, Silk JD, Ichiryu N, Davies TJ, Nolan KF, Leishman AJ, Carpenter L, Watt SM, Cerundolo V, Fairchild PJ. 2012. Cross-presentation of tumour antigens by human induced pluripotent stem cell-derived CD141 XCR1 dendritic cells Gene Therapy, 19 (10), pp. 1035-1040. | Show Abstract | Read more

Monocyte-derived dendritic cells (moDC) have been widely used in cancer immunotherapy but show significant donor-to-donor variability and low capacity for the cross-presentation of tumour-associated antigens (TAA) to CD8 T cells, greatly limiting the success of this approach. Given recent developments in induced pluripotency and the relative ease with which induced pluripotent stem (iPS) cell lines may be generated from individuals, we have succeeded in differentiating dendritic cells (DC) from human leukocyte antigen (HLA)-A 0201 iPS cells (iPS cell-derived DC (ipDC)), using protocols compliant with their subsequent clinical application. Unlike moDC, a subset of ipDC was found to coexpress CD141 and XCR1 that have been shown previously to define the human equivalent of mouse CD8α DC, in which the capacity for cross-presentation has been shown to reside. Accordingly, ipDC were able to cross-present the TAA, Melan A, to a CD8 T-cell clone and stimulate primary Melan A-specific responses among naïve T cells from an HLA-A 0201 donor. Given that CD141 XCR1 DC are present in peripheral blood in trace numbers that preclude their clinical application, the ability to generate a potentially unlimited source from iPS cells offers the possibility of harnessing their capacity for cross-priming of cytotoxic T lymphocytes for the induction of tumour-specific immune responses. © 2012 Macmillan Publishers Limited All rights reserved.

Chang PP, Barral P, Fitch J, Pratama A, Ma CS, Kallies A, Hogan JJ, Cerundolo V, Tangye SG, Bittman R et al. 2011. Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat Immunol, 13 (1), pp. 35-43. | Show Abstract | Read more

Lipid antigens trigger help from natural killer T cells (NKT cells) for B cells, and direct conjugation of lipid agonists to antigen profoundly augments antibody responses. Here we show that in vivo, NKT cells engaged in stable and prolonged cognate interactions with B cells and induced the formation of early germinal centers. Mouse and human NKT cells formed CXCR5(+)PD-1(hi) follicular helper NKT cells (NKT(FH) cells), and this process required expression of the transcriptional repressor Bcl-6, signaling via the coreceptor CD28 and interaction with B cells. NKT(FH) cells provided direct cognate help to antigen-specific B cells that was dependent on interleukin 21 (IL-21). Unlike T cell-dependent germinal centers, those driven by NKT(FH) cells did not generate long-lived plasma cells. Our results demonstrate the existence of a Bcl-6-dependent subset of NKT cells specialized in providing help to B cells.

Cited:

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Scopus

Davis LC, Morgan AJ, Chen JL, Snead CM, Bloor-Young D, Shenderov E, Stanton-Humphreys MN, Conway SJ, Churchill GC, Parrington J et al. 2012. NAADP Activates two-pore channels on t cell cytolytic granules to stimulate exocytosis and killing Current Biology, 22 (24), pp. 2331-2337. | Show Abstract | Read more

A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1-4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5-7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca 2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts. © 2012 Elsevier Ltd.

Jervis PJ, Moulis M, Jukes JP, Ghadbane H, Cox LR, Cerundolo V, Besra GS. 2012. Towards multivalent CD1d ligands: Synthesis and biological activity of homodimeric α-galactosyl ceramide analogues Carbohydrate Research, 356 pp. 152-162. | Show Abstract | Read more

A library of dimeric CD1d ligands, containing two α-galactosyl ceramide (α-GalCer) units linked by spacers of varying lengths has been synthesised. The key dimerisation reactions were carried out via copper-catalysed click reactions between a 6″-azido-6″-deoxy- α-galactosyl ceramide derivative and various diynes. Each α-GalCer dimer was tested for its ability to stimulate iNKT cells. © 2012 Elsevier Ltd. All rights reserved.

Silk KM, Silk JD, Ichiryu N, Davies TJ, Nolan KF, Leishman AJ, Carpenter L, Watt SM, Cerundolo V, Fairchild PJ. 2012. Cross-presentation of tumour antigens by human induced pluripotent stem cell-derived CD141(+)XCR1+ dendritic cells. Gene Ther, 19 (10), pp. 1035-1040. | Show Abstract | Read more

Monocyte-derived dendritic cells (moDC) have been widely used in cancer immunotherapy but show significant donor-to-donor variability and low capacity for the cross-presentation of tumour-associated antigens (TAA) to CD8(+) T cells, greatly limiting the success of this approach. Given recent developments in induced pluripotency and the relative ease with which induced pluripotent stem (iPS) cell lines may be generated from individuals, we have succeeded in differentiating dendritic cells (DC) from human leukocyte antigen (HLA)-A(*)0201(+) iPS cells (iPS cell-derived DC (ipDC)), using protocols compliant with their subsequent clinical application. Unlike moDC, a subset of ipDC was found to coexpress CD141 and XCR1 that have been shown previously to define the human equivalent of mouse CD8α(+) DC, in which the capacity for cross-presentation has been shown to reside. Accordingly, ipDC were able to cross-present the TAA, Melan A, to a CD8(+) T-cell clone and stimulate primary Melan A-specific responses among naïve T cells from an HLA-A(*)0201(+) donor. Given that CD141(+)XCR1(+) DC are present in peripheral blood in trace numbers that preclude their clinical application, the ability to generate a potentially unlimited source from iPS cells offers the possibility of harnessing their capacity for cross-priming of cytotoxic T lymphocytes for the induction of tumour-specific immune responses.

Silk JD, Lakhal S, Laynes R, Vallius L, Karydis I, Marcea C, Boyd CA, Cerundolo V. 2011. IDO induces expression of a novel tryptophan transporter in mouse and human tumor cells. J Immunol, 187 (4), pp. 1617-1625. | Show Abstract | Read more

IDO is the rate-limiting enzyme in the kynurenine pathway, catabolizing tryptophan to kynurenine. Tryptophan depletion by IDO-expressing tumors is a common mechanism of immune evasion inducing regulatory T cells and inhibiting effector T cells. Because mammalian cells cannot synthesize tryptophan, it remains unclear how IDO(+) tumor cells overcome the detrimental effects of local tryptophan depletion. We demonstrate that IDO(+) tumor cells express a novel amino acid transporter, which accounts for ∼50% of the tryptophan uptake. The induced transporter is biochemically distinguished from the constitutively expressed tryptophan transporter System L by increased resistance to inhibitors of System L, resistance to inhibition by high concentrations of most amino acids tested, and high substrate specificity for tryptophan. Under conditions of low extracellular tryptophan, expression of this novel transporter significantly increases tryptophan entry into IDO(+) tumors relative to tryptophan uptake through the low-affinity System L alone, and further decreases tryptophan levels in the microenvironment. Targeting this additional tryptophan transporter could be a way of pharmacological inhibition of IDO-mediated tumor escape. These findings highlight the ability of IDO-expressing tumor cells to thrive in a tryptophan-depleted microenvironment by expressing a novel, highly tryptophan-specific transporter, which is resistant to inhibition by most other amino acids. The additional transporter allows tumor cells to strike the ideal balance between supply of tryptophan essential for their own proliferation and survival, and depleting the extracellular milieu of tryptophan to inhibit T cell proliferation.

Stinchcombe JC, Salio M, Cerundolo V, Pende D, Arico M, Griffiths GM. 2011. Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems. BMC Biol, 9 (1), pp. 45. | Show Abstract | Read more

BACKGROUND: Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. RESULTS: NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. CONCLUSIONS: These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.

Bozna BL, Polzella P, Rankl C, Zhu R, Salio M, Shepherd D, Duman M, Cerundolo V, Hinterdorfer P. 2011. Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy. J Biol Chem, 286 (18), pp. 15973-15979. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

Stock A, Booth S, Cerundolo V. 2011. Prostaglandin E2 suppresses the differentiation of retinoic acid-producing dendritic cells in mice and humans. J Exp Med, 208 (4), pp. 761-773. | Show Abstract | Read more

The production of retinoic acid (RA) by dendritic cells (DCs) is critical for the induction of gut-tropic immune responses by driving the expression of intestinal-specific homing receptors, such as α4β7 and CCR9, upon T and B cell activation. However, how RA production is regulated during DC development remains unclear. We describe an unexpected role for prostaglandin E2 (PGE2) as a negative regulator of retinal dehydrogenases (RALDH), the enzymes responsible for RA synthesis. The presence of PGE2 during DC differentiation inhibited RALDH expression in mouse and human DCs, abrogating their ability to induce CCR9 expression upon T cell priming. Furthermore, blocking PGE2 signaling increased the frequency of RALDH(+) DCs in vitro, and reducing PGE2 synthesis in vivo promoted the systemic emergence of RA-producing DCs and the priming of CCR9(+) T cells in nonintestinal sites such as the spleen. Finally, we found that PGE2 stimulated the expression of the inducible cyclic AMP early repressor, which appears to directly inhibit RALDH expression in DCs, thus providing mechanistic insight into how PGE2 signaling down-modulates RALDH. Given the role of PGE2 in regulating the development of RA-producing DCs, modulating this pathway may prove a novel means to control the development of gut-tropic immune responses.

Hutchinson S, Sims S, O'Hara G, Silk J, Gileadi U, Cerundolo V, Klenerman P. 2011. A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus. PLoS One, 6 (2), pp. e14646. | Show Abstract | Read more

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8(+) T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8(+) T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8(+) T cell responses to MCMV - both conventional memory responses and those undergoing long-term expansion or "inflation". We infected LMP7(-/-) and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8(+) T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory "inflating" epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8(+) T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.

Dushek O, Aleksic M, Wheeler RJ, Zhang H, Cordoba SP, Peng YC, Chen JL, Cerundolo V, Dong T, Coombs D, van der Merwe PA. 2011. Antigen potency and maximal efficacy reveal a mechanism of efficient T cell activation. Sci Signal, 4 (176), pp. ra39. | Show Abstract | Read more

T cell activation, a critical event in adaptive immune responses, depends on productive interactions between T cell receptors (TCRs) and antigens presented as peptide-bound major histocompatibility complexes (pMHCs). Activated T cells lyse infected cells, secrete cytokines, and perform other effector functions with various efficiencies, which depend on the binding parameters of the TCR-pMHC complex. The mechanism through which binding parameters are translated to the efficiency of T cell activation, however, remains controversial. The "affinity model" suggests that the dissociation constant (KD) of the TCR-pMHC complex determines the response, whereas the "productive hit rate model" suggests that the off-rate (koff) is critical. Here, we used mathematical modeling to show that antigen potency, as determined by the EC50 (half-maximal effective concentration), which is used to support KD-based models, could not discriminate between the affinity and the productive hit rate models. Both models predicted a correlation between EC50 and KD, but only the productive hit rate model predicted a correlation between maximal efficacy (Emax), the maximal T cell response induced by pMHC, and koff. We confirmed the predictions made by the productive hit rate model in experiments with cytotoxic T cell clones and a panel of pMHC variants. Thus, we propose that the activity of an antigen is determined by both its potency (EC50) and maximal efficacy (Emax).

De Santo C, Salio M, Dong T, Reiter Y, Cerundolo V. 2011. Cerundolo et al. respond Nature Immunology, 12 (11), pp. 1018-1020. | Read more

Huang S, Cheng TY, Young DC, Layre E, Madigan CA, Shires J, Cerundolo V, Altman JD, Moody DB. 2011. Discovery of deoxyceramides and diacylglycerols as CD1b scaffold lipids among diverse groove-blocking lipids of the human CD1 system. Proc Natl Acad Sci U S A, 108 (48), pp. 19335-19340. | Show Abstract | Read more

Unlike the dominant role of one class II invariant chain peptide (CLIP) in blocking MHC class II, comparative lipidomics analysis shows that human cluster of differentiation (CD) proteins CD1a, CD1b, CD1c, and CD1d bind lipids corresponding to hundreds of diverse accurate mass retention time values. Although most ions were observed in association with several CD1 proteins, ligands binding selectively to one CD1 isoform allowed the study of how differing antigen-binding grooves influence lipid capture. Although the CD1b groove is distinguished by its unusually large volume (2,200 Å(3)) and the T' tunnel, the average mass of compounds eluted from CD1b was similar to that of lipids from CD1 proteins with smaller grooves. Elution of small ligands from the large CD1b groove might be explained if two small lipids bind simultaneously in the groove. Crystal structures indicate that all CD1 proteins can capture one antigen with its hydrophilic head group exposed for T-cell recognition, but CD1b structures show scaffold lipids seated below the antigen. We found that ligands selectively associated with CD1b lacked the hydrophilic head group that is generally needed for antigen recognition but interferes with scaffold function. Furthermore, we identified the scaffolds as deoxyceramides and diacylglycerols and directly demonstrate a function in augmenting presentation of a small glycolipid antigen to T cells. Thus, unlike MHC class II, CD1 proteins capture highly diverse ligands in the secretory pathway. CD1b has a mechanism for presenting either two small or one large lipid, allowing presentation of antigens with an unusually broad range of chain lengths.

Pei B, Speak AO, Shepherd D, Butters T, Cerundolo V, Platt FM, Kronenberg M. 2011. Diverse endogenous antigens for mouse NKT cells: self-antigens that are not glycosphingolipids. J Immunol, 186 (3), pp. 1348-1360. | Show Abstract | Read more

NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.

Kaur R, Chen J, Dawoodji A, Cerundolo V, Garcia-Diaz YR, Wojno J, Cox LR, Besra GS, Moghaddam B, Perrie Y. 2011. Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells. J Pharm Sci, 100 (7), pp. 2724-2733. | Show Abstract | Read more

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid α-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-γ) and interleukin-4 (IL-4). However, αGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the αGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-γ secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Kaur R, Chen J, Dawoodji A, Cerundolo V, Garcia-Diaz YR, Wojno J, Cox LR, Besra GS, Moghaddam B, Perrie Y. 2011. Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells Journal of Pharmaceutical Sciences, 100 (7), pp. 2724-2733. | Show Abstract | Read more

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid α-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-γ) and interleukin-4 (IL-4). However, αGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the αGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-γ secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association.

De Santo C, Salio M, Dong T, Reiter Y, Cerundolo V. 2011. Reply to "Failure to detect production of IL-10 by activated human neutrophils". Nat Immunol, 12 (11), pp. 1018-1020. | Read more

Veerapen N, Reddington F, Salio M, Cerundolo V, Besra GS. 2011. Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation Bioorganic and Medicinal Chemistry, 19 (1), pp. 221-228. | Show Abstract | Read more

Stimulation of iNKT cells by α-galactosyl ceramide (α-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells' response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in α-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of α-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation. © 2010 Elsevier Ltd. All rights reserved.

De Santo C, Arscott R, Booth S, Karydis I, Jones M, Asher R, Salio M, Middleton M, Cerundolo V. 2010. Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A. Nat Immunol, 11 (11), pp. 1039-1046. | Show Abstract | Read more

Neutrophils are the main effector cells during inflammation, but they can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms that modulate their plasticity remain unclear. We now show that systemic serum amyloid A 1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory interleukin 10 (IL-10)-secreting neutrophils but also promoted the interaction of invariant natural killer T cells (iNKT cells) with those neutrophils, a process that limited their suppressive activity by diminishing the production of IL-10 and enhancing the production of IL-12. Because SAA-1-producing melanomas promoted differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses.

Wojno J, Jukes J-P, Polzella P, Cerundolo V, Cox LR, Besra GS. 2010. Novel glycolipids in CD1d-mediated immunity: Synthesis of new agonists for CD1d ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 240

Zeissig S, Dougan SK, Barral DC, Junker Y, Chen Z, Kaser A, Ho M, Mandel H, McIntyre A, Kennedy SM et al. 2010. Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function. J Clin Invest, 120 (8), pp. 2889-2899. | Show Abstract | Read more

Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

Nika K, Soldani C, Salek M, Paster W, Gray A, Etzensperger R, Fugger L, Polzella P, Cerundolo V, Dushek O et al. 2010. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity, 32 (6), pp. 766-777. | Show Abstract | Read more

T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

Poulin LF, Salio M, Griessinger E, Anjos-Afonso F, Craciun L, Chen JL, Keller AM, Joffre O, Zelenay S, Nye E et al. 2010. Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells. J Exp Med, 207 (6), pp. 1261-1271. | Show Abstract | Read more

In mouse, a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

Cerundolo V, Barral P, Batista FD. 2010. Synthetic iNKT cell-agonists as vaccine adjuvants--finding the balance. Curr Opin Immunol, 22 (3), pp. 417-424. | Show Abstract | Read more

The unique position of invariant natural killer T (iNKT) cells at the interface of the innate and adaptive arms of the immune response, combined with their ability to modulate the activity of antigen-presenting cells, has led to their intensive investigation as a means of augmenting the immune response both in vaccination strategies for microbial infections and in tumor immunotherapy. Several synthetic iNKT-cell agonists that have potential as vaccine adjuvants have been identified, but these are not without their limitations-strong agonists can lead to the undesirable effects associated with overstimulation of the immune system, whereas too weak agonists may provide insufficient iNKT cell help to stimulate maturation of dendritic cells and differentiation of B cells. In this article we explore strategies being investigated as means of increasing the specificity of and controlling the magnitude of the immune response generated by activation of iNKT cells with synthetic agonists.

Barral P, Polzella P, Bruckbauer A, van Rooijen N, Besra GS, Cerundolo V, Batista FD. 2010. CD169(+) macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes. Nat Immunol, 11 (4), pp. 303-312. | Show Abstract | Read more

Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169(+) macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169(+) macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses.

Duman M, Pfleger M, Zhu R, Rankl C, Chtcheglova LA, Neundlinger I, Bozna BL, Mayer B, Salio M, Shepherd D et al. 2010. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging. Nanotechnology, 21 (11), pp. 115504. | Show Abstract | Read more

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Aleksic M, Dushek O, Zhang H, Shenderov E, Chen JL, Cerundolo V, Coombs D, van der Merwe PA. 2010. Dependence of T cell antigen recognition on T cell receptor-peptide MHC confinement time. Immunity, 32 (2), pp. 163-174. | Show Abstract | Read more

T cell receptor (TCR) binding to diverse peptide-major histocompatibility complex (pMHC) ligands results in various degrees of T cell activation. Here we analyze which binding properties of the TCR-pMHC interaction are responsible for this variation in pMHC activation potency. We have analyzed activation of the 1G4 cytotoxic T lymphocyte clone by cognate pMHC variants and performed thorough correlation analysis of T cell activation with 1G4 TCR-pMHC binding properties measured in solution. We found that both the on rate (k(on)) and off rate (k(off)) contribute to activation potency. Based on our results, we propose a model in which rapid TCR rebinding to the same pMHC after chemical dissociation increases the effective half-life or "confinement time" of a TCR-pMHC interaction. This confinement time model clarifies the role of k(on) in T cell activation and reconciles apparently contradictory reports on the role of TCR-pMHC binding kinetics and affinity in T cell activation.

Cerundolo V, Kronenberg M. 2010. The role of invariant NKT cells at the interface of innate and adaptive immunity. Semin Immunol, 22 (2), pp. 59-60. | Read more

Salio M, Silk JD, Cerundolo V. 2010. Recent advances in processing and presentation of CD1 bound lipid antigens. Curr Opin Immunol, 22 (1), pp. 81-88. | Show Abstract | Read more

It is well established that different populations of alphabeta T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules, but also foreign and self-lipids in association with CD1 proteins, which share structural similarities with MHC class I molecules. CD1 molecules are comprised of five isoforms, known as group 1 (CD1a, b, c, e) and group 2 (CD1d) CD1, presenting lipid antigens to conventional T lymphocytes or innate-like T cells bearing an invariant T cell receptor (TCR) and known as invariant NKT (iNKT) cells. During the last couple of years, several papers have been published describing important aspects of the mechanisms controlling the processing and presentation of endogenous and exogenous CD1 lipid antigens, which will be the main focus of this review.

Röhrig UF, Awad L, Grosdidier A, Larrieu P, Stroobant V, Colau D, Cerundolo V, Simpson AJ, Vogel P, Van den Eynde BJ et al. 2010. Rational design of indoleamine 2,3-dioxygenase inhibitors. J Med Chem, 53 (3), pp. 1172-1189. | Show Abstract | Read more

Indoleamine 2,3-dioxygenase (IDO) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. We have used the evolutionary docking algorithm EADock to design new inhibitors of this enzyme. First, we investigated the modes of binding of all known IDO inhibitors. On the basis of the observed docked conformations, we developed a pharmacophore model, which was then used to devise new compounds to be tested for IDO inhibition. We also used a fragment-based approach to design and to optimize small organic molecule inhibitors. Both approaches yielded several new low-molecular weight inhibitor scaffolds, the most active being of nanomolar potency in an enzymatic assay. Cellular assays confirmed the potential biological relevance of four different scaffolds.

Chen JL, Morgan AJ, Stewart-Jones G, Shepherd D, Bossi G, Wooldridge L, Hutchinson SL, Sewell AK, Griffiths GM, van der Merwe PA et al. 2010. Ca2+ release from the endoplasmic reticulum of NY-ESO-1-specific T cells is modulated by the affinity of TCR and by the use of the CD8 coreceptor. J Immunol, 184 (4), pp. 1829-1839. | Show Abstract | Read more

Although several cancer immunotherapy strategies are based on the use of analog peptides and on the modulation of the TCR affinity of adoptively transferred T cells, it remains unclear whether tumor-specific T cell activation by strong and weak TCR stimuli evoke different Ca(2+) signatures from the Ca(2+) intracellular stores and whether the amplitude of Ca(2+) release from the endoplasmic reticulum (ER) can be further modulated by coreceptor binding to peptide/MHC. In this study, we combined functional, structural, and kinetic measurements to correlate the intensity of Ca(2+) signals triggered by the stimulation of the 1G4 T cell clone specific to the tumor epitope NY-ESO-1(157-165). Two analogs of the NY-ESO-1(157-165) peptide, having similar affinity to HLA-A2 molecules, but a 6-fold difference in binding affinity for the 1G4 TCR, resulted in different Ca(2+) signals and T cell activation. 1G4 stimulation by the stronger stimulus emptied the ER of stored Ca(2+), even in the absence of CD8 binding, resulting in sustained Ca(2+) influx. In contrast, the weaker stimulus induced only partial emptying of stored Ca(2+), resulting in significantly diminished and oscillatory Ca(2+) signals, which were enhanced by CD8 binding. Our data define the range of TCR/peptide MHC affinities required to induce depletion of Ca(2+) from intracellular stores and provide insights into the ability of T cells to tailor the use of the CD8 coreceptor to enhance Ca(2+) release from the ER. This, in turn, modulates Ca(2+) influx from the extracellular environment, ultimately controlling T cell activation.

Wadle A, Mischo A, Strahl S, Nishikawa H, Held G, Neumann F, Wullner B, Fischer E, Kleber S, Karbach J et al. 2010. NY-ESO-1 protein glycosylated by yeast induces enhanced immune responses. Yeast, 27 (11), pp. 919-931. | Show Abstract | Read more

Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full-length tumour-associated antigen NY-ESO-1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY-ESO-1 protein linked to the yeast a-agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross-presentation of NY-ESO-1-derived peptides. The process of antigen uptake and cross-presentation was dependent on the glycosylation pattern of NY-ESO-1-Aga2p protein and the presence of accessible mannose receptors. In addition, NY-ESO-1-Aga2p protein uptake by dendritic cells resulted in recognition by HLA-DP4 NY-ESO-1-specific CD4(+) T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast-derived NY-ESO-1-Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY-ESO-1 protein. Together, these data demonstrate that yeast-derived full-length NY-ESO-1-Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development.

Gnjatic S, Cao Y, Reichelt U, Yekebas EF, Nölker C, Marx AH, Erbersdobler A, Nishikawa H, Hildebrandt Y, Bartels K et al. 2010. NY-CO-58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer. Int J Cancer, 127 (2), pp. 381-393. | Show Abstract | Read more

NY-CO-58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY-CO-58/KIF2C by RT-PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full-length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY-CO-58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki-67 expression (r = 0.69; p = 0.0003). Investigating NY-CO-58/KIF2C-specific T cell responses, CD8(+) T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4(+) T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN-gamma and TNF-alpha. Depletion of CD4(+)CD25(+) T cells before stimulation significantly increased the intensity of the preexisting response. NY-CO-58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY-CO-58/KIF2C was able to induce spontaneous CD4(+) T cell responses of the Th1-type, which were tightly controlled by peripheral T regulatory cells.

Salio M, Cerundolo V. 2009. Linking inflammation to natural killer T cell activation. PLoS Biol, 7 (10), pp. e1000226. | Show Abstract | Read more

Immune activation is often associated with inflammation, but inflammation's role in the expansion of antigen-specific immune responses remains unclear. This primer focuses on recent findings that show how specific natural killer T cells are activated by inflammatory messengers, thus illuminating the cellular and molecular links between immunity and inflammation.

Ishizuka J, Grebe K, Shenderov E, Peters B, Chen Q, Peng Y, Wang L, Dong T, Pasquetto V, Oseroff C et al. 2009. Quantitating T cell cross-reactivity for unrelated peptide antigens. J Immunol, 183 (7), pp. 4337-4345. | Show Abstract | Read more

Quantitating the frequency of T cell cross-reactivity to unrelated peptides is essential to understanding T cell responses in infectious and autoimmune diseases. Here we used 15 mouse or human CD8+ T cell clones (11 antiviral, 4 anti-self) in conjunction with a large library of defined synthetic peptides to examine nearly 30,000 TCR-peptide MHC class I interactions for cross-reactions. We identified a single cross-reaction consisting of an anti-self TCR recognizing a poxvirus peptide at relatively low sensitivity. We failed to identify any cross-reactions between the synthetic peptides in the panel and polyclonal CD8+ T cells raised to viral or alloantigens. These findings provide the best estimate to date of the frequency of T cell cross-reactivity to unrelated peptides ( approximately 1/30,000), explaining why cross-reactions between unrelated pathogens are infrequently encountered and providing a critical parameter for understanding the scope of self-tolerance.

Lawson VJ, Maurice D, Silk JD, Cerundolo V, Weston K. 2009. Aberrant selection and function of invariant NKT cells in the absence of AP-1 transcription factor Fra-2. J Immunol, 183 (4), pp. 2575-2584. | Show Abstract | Read more

The transcription factors mediating the development of CD1d-restricted invariant NKT (iNKT) cells remain incompletely described. Here, we show that loss of the AP-1 transcription factor Fra-2 causes a marked increase in the number of both thymic and peripheral iNKT cells, without affecting the development of other T-lineage cells. The defect is cell-autonomous and is evident in the earliest iNKT precursors. We find that iNKT cells expressing the lower affinity TCRVbeta8 are proportionally overrepresented in the absence of Fra-2, indicating altered selection of iNKT cells. There is also widespread dysregulation of AP-1-directed gene expression. In the periphery, mature Fra-2-deficient iNKT cells are able to participate in an immune response, but they have an altered response to Ag, showing increased expansion and producing increased amounts of IL-2 and IL-4 compared with their wild-type counterparts. Unusually, naive Fra-2-deficient T cells also rapidly produce IL-2 and IL-4 upon activation. Taken together, these data define Fra-2 as necessary for regulation of normal iNKT cell development and function, and they demonstrate the central role played by the AP-1 family in this lineage.

Veerapen N, Brigl M, Garg S, Cerundolo V, Cox LR, Brenner MB, Besra GS. 2009. Synthesis and biological activity of alpha-galactosyl ceramide KRN7000 and galactosyl (alpha1-->2) galactosyl ceramide. Bioorg Med Chem Lett, 19 (15), pp. 4288-4291. | Show Abstract | Read more

We herein report a faster and less cumbersome synthesis of the biologically attractive, alpha-galactosyl ceramide (alpha-GalCer), known as KRN7000, and its analogues. More importantly, the use of a silicon tethered intramolecular glycosylation reaction gave easy access to the diglycosyl ceramide Gal(alpha1-->2)GalCer, which has been shown to require uptake and processing to the biologically active alpha-GalCer derivative.

Mallevaey T, Scott-Browne JP, Matsuda JL, Young MH, Pellicci DG, Patel O, Thakur M, Kjer-Nielsen L, Richardson SK, Cerundolo V et al. 2009. T cell receptor CDR2 beta and CDR3 beta loops collaborate functionally to shape the iNKT cell repertoire. Immunity, 31 (1), pp. 60-71. | Show Abstract | Read more

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S et al. 2009. Rational development of high-affinity T-cell receptor-like antibodies (vol 106, pg 5784, 2009) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106 (26), pp. 10872-10872. | Read more

Palmowski MJ, Parker M, Choudhuri K, Chiu C, Callan MF, van der Merwe PA, Cerundolo V, Gould KG. 2009. A single-chain H-2Db molecule presenting an influenza virus nucleoprotein epitope shows enhanced ability at stimulating CD8+ T cell responses in vivo. J Immunol, 182 (8), pp. 4565-4571. | Show Abstract | Read more

We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.

Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S et al. 2009. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci U S A, 106 (14), pp. 5784-5788. | Show Abstract | Read more

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

Reddy BG, Silk JD, Salio M, Balamurugan R, Shepherd D, Ritter G, Cerundolo V, Schmidt RR. 2009. Nonglycosidic agonists of invariant NKT cells for use as vaccine adjuvants. ChemMedChem, 4 (2), pp. 171-175. | Show Abstract | Read more

Based on the crystal structures of human alpha-GalCer-CD1d and iNKT-alpha-GalCer-CD1d complexes, nonglycosidic analogues of alpha-GalCer were synthesized. They activate iNKT cells resulting in dendritic cell maturation and the priming of antigen-specific T and B cells. Therefore, they are attractive adjuvants in vaccination strategies for cancer and infectious diseases.

Stock A, Cerundolo V. 2009. Analysis of frequency and phenotype of antigen-specific T cells. Methods Mol Biol, 514 pp. 1-14. | Show Abstract | Read more

Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells and for carrying out ICS to measure antigen-specific T lymphocytes.

Cerundolo V, Silk JD, Masri SH, Salio M. 2009. Harnessing invariant NKT cells in vaccination strategies. Nat Rev Immunol, 9 (1), pp. 28-38. | Show Abstract | Read more

To optimize vaccination strategies, it is important to use protocols that can 'jump-start' immune responses by harnessing cells of the innate immune system to assist the expansion of antigen-specific B and T cells. In this Review, we discuss the evidence indicating that invariant natural killer T (iNKT) cells can positively modulate dendritic cells and B cells, and that their pharmacological activation in the presence of antigenic proteins can enhance antigen-specific B- and T-cell responses. In addition, we describe structural and kinetic analyses that assist in the design of optimal iNKT-cell agonists that could be used in the clinical setting as vaccine adjuvants.

Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S et al. 2009. Correction for Stewart-Jones et al., Rational development of high-affinity T-cell receptor-like antibodies Proceedings of the National Academy of Sciences, 106 (26), pp. 10872-10872. | Read more

De Santo C, Salio M, Masri SH, Lee LY, Dong T, Speak AO, Porubsky S, Booth S, Veerapen N, Besra GS et al. 2008. Invariant NKT cells reduce the immunosuppressive activity of influenza A virus-induced myeloid-derived suppressor cells in mice and humans. J Clin Invest, 118 (12), pp. 4036-4048. | Show Abstract | Read more

Infection with influenza A virus (IAV) presents a substantial threat to public health worldwide, with young, elderly, and immunodeficient individuals being particularly susceptible. Inflammatory responses play an important role in the fatal outcome of IAV infection, but the mechanism remains unclear. We demonstrate here that the absence of invariant NKT (iNKT) cells in mice during IAV infection resulted in the expansion of myeloid-derived suppressor cells (MDSCs), which suppressed IAV-specific immune responses through the expression of both arginase and NOS, resulting in high IAV titer and increased mortality. Adoptive transfer of iNKT cells abolished the suppressive activity of MDSCs, restored IAV-specific immune responses, reduced IAV titer, and increased survival rate. The crosstalk between iNKT and MDSCs was CD1d- and CD40-dependent. Furthermore, IAV infection and exposure to TLR agonists relieved the suppressive activity of MDSCs. Finally, we extended these results to humans by demonstrating the presence of myeloid cells with suppressive activity in the PBLs of individuals infected with IAV and showed that their suppressive activity is substantially reduced by iNKT cell activation. These findings identify what we believe to be a novel immunomodulatory role of iNKT cells, which we suggest could be harnessed to abolish the immunosuppressive activity of MDSCs during IAV infection.

Porubsky S, Luckow B, Bonrouhi M, Speak A, Cerundolo V, Platt F, Gröne HJ. 2008. [Glycosphingolipids Gb3 and iGb3. In vivo roles in hemolytic-uremic syndrome and iNKT cell function]. Pathologe, 29 Suppl 2 (S2), pp. 297-302. | Show Abstract | Read more

UNLABELLED: The glycosphingolipids globotrihexosylceramide (Gb3, CD77) and isoglobotrihexosylceramide (iGb3) are isomers differing only in one glycosidic bond and have been implicated in several processes of the innate and adaptive immune system. AIMS: 1) To verify the function of Gb3 in the pathogenesis of hemolytic-uremic syndrome as the cellular receptor responsible for cytotoxicity caused by verotoxin (VT) elaborated by Shigella and certain strains of E.coli. 2) To investigate in vivo the previously implicated function of iGb3 as the endogenous lipid ligand responsible for positive selection of invariant natural killer T-cells (iNKT), which have an essential regulatory function in infection, tumor rejection and tolerance. METHODS: Generation of mice deficient in Gb3 and iGb3 synthesizing enzymes and VT injection into Gb3-deficient mice. Analysis of iNKT cell development and function by flow cytometry and by administration of the exogenous agonist alpha-galactosylceramide in iGb3-deficient mice. RESULTS: For 1) Gb3-deficient mice were insensitive to otherwise lethal doses of VT, and 2) iGb3-deficient mice showed normal numbers of iNKT cells. Furthermore the function of iNKT cells evolving in iGb3-deficient mice was unaffected. CONCLUSIONS: 1) Gb3 is the cellular receptor mediating verotoxin cytotoxicity in haemolytic-uremic syndrome. 2) In contrast to previous indirect implications, iGb3 cannot be regarded as an endogenous ligand responsible for the positive selection of iNKT cells.

Speak AO, Cerundolo V, Platt FM. 2008. CD1d presentation of glycolipids. Immunol Cell Biol, 86 (7), pp. 588-597. | Show Abstract | Read more

The CD1 family of antigen-presenting molecules consists of five members, CD1a to e. Of these molecules CD1d has been the subject of much interest over the past 10 years following the discovery that this molecule presents antigens to a group of T cells known as invariant natural killer T cells (iNKT). iNKT cells carry an invariant T cell receptor which contains homologous gene segments in mouse and man. iNKT cells are positively selected in the thymus in the same manner as major histocompatibility complex restricted T cells, except iNKT cells require CD1d to be presented by thymocytes rather than epithelial cells. Once in peripheral organs, iNKT cells appear to play multiple roles in host defence against pathogens and cancer. If the numbers of iNKT cells are not correctly regulated it can result in autoimmune disorders, such as diabetes. The ligands for iNKT cells have been the subject of much research but identifying physiologically relevant candidate ligands for positive selection or activation has proved technically very challenging. This is largely due to the fact that the ligands for iNKT cells are lipids. The lipid ligands for thymic selection and some of those involved in peripheral activation are self-derived. Glycosphingolipids are suggested to be the class of lipid for iNKT cell thymic development. For peripheral activation it appears multiple classes of self-derived lipids may play a role, in addition to pathogen-derived lipids. This review will cover essential background to iNKT cell and CD1d biology with emphasis on the candidate iNKT cell ligands proposed to date.

Barral P, Eckl-Dorna J, Harwood NE, De Santo C, Salio M, Illarionov P, Besra GS, Cerundolo V, Batista FD. 2008. B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo. Proc Natl Acad Sci U S A, 105 (24), pp. 8345-8350. | Show Abstract | Read more

Highly regulated activation of B cells is required for the production of specific antibodies necessary to provide protection from pathogen infection. This process is initiated by specific recognition of antigen through the B cell receptor (BCR), leading to early intracellular signaling followed by the late recruitment of T cell help. In this study we demonstrate that specific BCR uptake of CD1d-restricted antigens represents an effective means of enhancing invariant natural killer T (iNKT)-dependent B cell responses in vivo. This mechanism is effective over a wide range of antigen affinities but depends on exceeding a tightly regulated avidity threshold necessary for BCR-mediated internalization and CD1d-dependent presentation of particulate antigenic lipid. Subsequently, iNKT cells provide the help required for stimulating B cell proliferation and differentiation. iNKT-stimulated B cells develop within extrafollicular foci and mediate the production of high titers of specific IgM and early class-switched antibodies. Thus, we have demonstrated that in response to particulate antigenic lipids iNKT cells are recruited for the assistance of B cell activation, resulting in the enhancement of specific antibody responses. We propose that such a mechanism may operate to potentiate adaptive immune responses against pathogens in vivo.

Silk JD, Salio M, Reddy BG, Shepherd D, Gileadi U, Brown J, Masri SH, Polzella P, Ritter G, Besra GS et al. 2008. Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells. J Immunol, 180 (10), pp. 6452-6456. | Show Abstract

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

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Silk JD, Salio M, Reddy BG, Shepherd D, Gileadi U, Brown J, Masri SH, Polzella P, Ritter G, Besra GS et al. 2008. Nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells JOURNAL OF IMMUNOLOGY, 180 (10), pp. 6452-6456.

Held G, Wadle A, Dauth N, Stewart-Jones G, Sturm C, Thiel M, Zwick C, Dieckmann D, Schuler G, Hoogenboom HR et al. 2008. MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing (vol 37, pg 2008, 2007) EUROPEAN JOURNAL OF IMMUNOLOGY, 38 (5), pp. 1465-1466. | Read more

Barnes E, Salio M, Cerundolo V, Francesco L, Pardoll D, Klenerman P, Cox A. 2008. Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus. J Viral Hepat, 15 (3), pp. 219-228. | Show Abstract | Read more

Studies assessing the function of monocyte derived dendritic cells (MD-DC) in individuals with hepatitis C virus (HCV) infection have shown conflicting results. Impaired MD-DC function in chronic HCV infection would have important implications both for understanding the pathogenesis of HCV infection and in the use of autologous MD-DC in vaccination strategies. We determined the allostimulatory capacity of MD-DC in the same patient before and after HCV infection. Next, the phenotype, cytokine production and allostimulatory function of immature and mature MD-DC in individuals with persistent HCV infection were compared directly with MD-DC from healthy individuals. Finally, we assessed the ability of MD-DC to prime autologous naïve peptide specific CD8+ T cells using HLA-A2 class-I tetramers. DCs retained the same allostimulatory capacity before and following the establishment of persistent HCV infection. The surface phenotype and the amount of interleukin (IL)-10 and IL-12(p70) produced during DC maturation did not differ between HCV-infected individuals and healthy controls. Mature DCs from HCV-infected individuals performed comparably in an allogeneic MLR compared with healthy individuals. Mature MD-DC from HCV-infected individuals stimulated the expansion of peptide specific naïve CD8+ T cells. MD-DC from HCV-infected and healthy individuals are phenotypically indistinguishable and perform comparably in functional assays.

Denkberg G, Stronge VS, Zahavi E, Pittoni P, Oren R, Shepherd D, Salio M, McCarthy C, Illarionov PA, van der Merwe A et al. 2008. Phage display-derived recombinant antibodies with TCR-like specificity against alpha-galactosylceramide and its analogues in complex with human CD1d molecules. Eur J Immunol, 38 (3), pp. 829-840. | Show Abstract | Read more

The glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of alpha-GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen-presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor-like (TCRL) specificity against the human CD1d (hCD1d)-alpha-GalCer complex. These Fab fragments were able to detect specifically hCD1d-alpha-GalCer complexes in cell-free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d(+) antigen-presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c-hCD1d-transgenic mice administered in vivo with alpha-GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of alpha-GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the alpha-GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of alpha-GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of alpha-GalCer and its analogues.

Thomas M, Boname JM, Field S, Nejentsev S, Salio M, Cerundolo V, Wills M, Lehner PJ. 2008. Down-regulation of NKG2D and NKp80 ligands by Kaposi's sarcoma-associated herpesvirus K5 protects against NK cell cytotoxicity. Proc Natl Acad Sci U S A, 105 (5), pp. 1656-1661. | Show Abstract | Read more

Natural killer (NK) cells are important early mediators of host immunity to viral infections. The NK activatory receptors NKG2D and NKp80, both C-type lectin-like homodimeric receptors, stimulate NK cell cytotoxicity toward target cells. Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) down-regulates MHC class I molecules to avoid detection by cytotoxic T lymphocytes but renders cells susceptible to NK cell cytotoxicity. We now show that the KSHV immune evasion gene, K5, reduces cell surface expression of the NKG2D ligands MHC class I-related chain A (MICA), MICB, and the newly defined ligand for NKp80, activation-induced C-type lectin (AICL). Down-regulation of both MICA and AICL requires the ubiquitin E3 ligase activity of K5 to target substrate cytoplasmic tail lysine residues. The common MICA *008 allele has a frameshift mutation leading to a premature stop codon and is resistant to down-regulation because of the loss of lysine residues. K5-mediated ubiquitylation signals internalization but not degradation of MICA and causes a potent reduction in NK cell-mediated cytotoxicity. The down-regulation of ligands for both the NKG2D and NKp80 activation pathways provides KSHV with a powerful mechanism for evasion of NK cell antiviral functions.

Porubsky S, Luckow B, Bonrouhi M, Speak A, Cerundolo V, Platt F, Gröne HJ. 2008. Glycosphingolipids Gb3 and iGb3. In vivo roles in hemolytic-uremic syndrome and iNKT cell function Pathologe, 29 (SUPPL. 2), pp. 297-302. | Show Abstract | Read more

The glycosphingolipids globotrihexosylceramide (Gb3, CD77) and isoglobotrihexosylceramide (iGb3) are isomers differing only in one glycosidic bond and have been implicated in several processes of the innate and adaptive immune system. Aims. 1) To verify the function of Gb3 in the pathogenesis of hemolytic-uremic syndrome as the cellular receptor responsible for cytotoxicity caused by verotoxin (VT) elaborated by Shigella and certain strains of E.coli. 2) To investigate in vivo the previously implicated function of iGb3 as the endogenous lipid ligand responsible for positive selection of invariant natural killer T-cells (iNKT), which have an essential regulatory function in infection, tumor rejection and tolerance. Methods. Generation of mice deficient in Gb3 and iGb3 synthesizing enzymes and VT injection into Gb3-deficient mice. Analysis of iNKT cell development and function by flow cytometry and by administration of the exogenous agonist alpha-galactosylceramide in iGb3-deficient mice. Results. For 1) Gb3-deficient mice were insensitive to otherwise lethal doses of VT, and 2) iGb3-deficient mice showed normal numbers of iNKT cells. Furthermore the function of iNKT cells evolving in iGb3-deficient mice was unaffected. Conclusions. 1) Gb3 is the cellular receptor mediating verotoxin cytotoxicity in haemolytic-uremic syndrome. 2) In contrast to previous indirect implications, iGb3 cannot be regarded as an endogenous ligand responsible for the positive selection of iNKT cells. © 2008 Springer Medizin Verlag.

Lopes L, Dewannieux M, Gileadi U, Bailey R, Ikeda Y, Whittaker C, Collin MP, Cerundolo V, Tomihari M, Ariizumi K, Collins MK. 2008. Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses. J Virol, 82 (1), pp. 86-95. | Show Abstract | Read more

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2(+) cells after intravenous injection and in CD11c(+) dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8(+) T-cell response in HLA-A2 transgenic mice and stimulated a CD4(+) T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A(b). As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.

Silk JD, Salio M, Brown J, Jones EY, Cerundolo V. 2008. Structural and functional aspects of lipid binding by CD1 molecules. Annu Rev Cell Dev Biol, 24 (1), pp. 369-395. | Show Abstract | Read more

Over the past ten years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I molecules the CD1 proteins. We describe the events that have led to the discovery of the role of CD1 molecules, their pattern of intracellular trafficking, and their ability to sample different intracellular compartments for self- and foreign lipids. Structural and functional aspects of lipid presentation by CD1 molecules are presented in the context of the function of CD1-restricted T cells in antimicrobial responses, antitumor immunity, and the regulation of the tolerance and autoimmunity immunoregulatory axis. Particular emphasis is on invariant NKT (iNKT) cells and their ability to modulate innate and adaptive immune responses.

Salio M, Speak AO, Shepherd D, Polzella P, Illarionov PA, Veerapen N, Besra GS, Platt FM, Cerundolo V. 2007. Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. Proc Natl Acad Sci U S A, 104 (51), pp. 20490-20495. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are a subset of nonconventional T cells recognizing endogenous and/or exogenous glycolipid antigens in the context of CD1d molecules. It remains unclear whether innate stimuli can modify the profile of endogenous lipids recognized by iNKT cells on the surface of antigen-presenting cells (APCs). We report that activation of human APCs by Toll-like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells, as defined by IFN-gamma secretion. APC-derived soluble factors further increase CD1d-restricted iNKT cell activation. Finally, using soluble tetrameric iNKT T cell receptors (TCR) as a staining reagent, we demonstrate specific up-regulation of CD1d-bound ligand(s) on TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of APCs resulting in iNKT cell activation and APC maturation underscores the role of iNKT cells in assisting priming of antigen-specific immune responses.

Koch M, Camp S, Collen T, Avila D, Salomonsen J, Wallny HJ, van Hateren A, Hunt L, Jacob JP, Johnston F et al. 2007. Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity, 27 (6), pp. 885-899. | Show Abstract | Read more

Little is known about the structure of major histocompatibility complex (MHC) molecules outside of mammals. Only one class I molecule in the chicken MHC is highly expressed, leading to strong genetic associations with infectious pathogens. Here, we report two structures of the MHC class I molecule BF2*2101 from the B21 haplotype, which is known to confer resistance to Marek's disease caused by an oncogenic herpesvirus. The binding groove has an unusually large central cavity, which confers substantial conformational flexibility to the crucial residue Arg9, allowing remodeling of key peptide-binding sites. The coupled variation of anchor residues from the peptide, utilizing a charge-transfer system unprecedented in MHC molecules, allows peptides with conspicuously different sequences to be bound. This promiscuous binding extends our understanding of ways in which MHC class I molecules can present peptides to the immune system and might explain the resistance of the B21 haplotype to Marek's disease.

Matthews KE, Qin JS, Yang J, Hermans IF, Palmowski MJ, Cerundolo V, Ronchese F. 2007. Increasing the survival of dendritic cells in vivo does not replace the requirement for CD4+ T cell help during primary CD8+ T cell responses. J Immunol, 179 (9), pp. 5738-5747. | Show Abstract

The survival of dendritic cells (DC) in vivo determines the duration of Ag presentation and is critical in determining the strength and magnitude of the resulting T cell response. We used a mouse model to show that Ag-loaded C57BL/6 DC (MHC class II(+/+) (MHC II(+/+))) that reach the lymph node survived longer than Ag-loaded MHC II(-/-) DC, with the numbers of C57BL/6 DC being approximately 2.5-fold the number of the MHC II(-/-) DC by day 4 and approximately 5-fold by day 7. The differential survival of DC in vivo was not affected by low doses of LPS, but in vitro pretreatment with CD40L or with high doses of LPS increased the numbers of MHC II(-/-) DC to levels approaching those of C57BL/6 DC. Regardless of their numbers and relative survival in lymph nodes, MHC II(-/-) DC were profoundly defective in their ability to induce CTL responses against the gp33 peptide epitope, and were unable to induce expansion and optimal cytotoxic activity of CD8(+) T cells specific for the male Ag UTY. We conclude that CD4(+) T cell help for CD8(+) responses involves mechanisms other than the increased survival of Ag-presenting DC in the lymph node.

Stronge VS, Salio M, Jones EY, Cerundolo V. 2007. A closer look at CD1d molecules: new horizons in studying NKT cells. Trends Immunol, 28 (10), pp. 455-462. | Show Abstract | Read more

Recent findings have highlighted the ability of invariant natural killer T (iNKT) cells to recognize microbe-derived glycolipids and have demonstrated the role of these cells in several disease states, from autoimmune disease to cancer. It has also become clear that iNKT cells can rapidly mature dendritic cells and licence them to prime antigen-specific T- and B-cell responses. The use of CD1d tetramers to monitor iNKT cell frequency and phenotype has moved the field forward at a fast pace. To harness iNKT cells for therapeutic purposes and to understand their role in vivo, it is essential to characterize the molecular events that contribute to iNKT cell activation. Here we review new reagents and novel protocols that are facilitating a closer look at lipid presentation by CD1d molecules and their recognition by iNKT cells.

Jones EY, Salio M, Cerundolo V. 2007. T cell receptors get back to basics. Nat Immunol, 8 (10), pp. 1033-1035. | Read more

Held G, Wadle A, Dauth N, Stewart-Jones G, Sturm C, Thiel M, Zwick C, Dieckmann D, Schuler G, Hoogenboom HR et al. 2007. MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing. Eur J Immunol, 37 (7), pp. 2008-2017. | Show Abstract | Read more

MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 1100-2600 [corrected] ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10- to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 16000-52000 [corrected] GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.

McCarthy C, Shepherd D, Fleire S, Stronge VS, Koch M, Illarionov PA, Bossi G, Salio M, Denkberg G, Reddington F et al. 2007. The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation. J Exp Med, 204 (5), pp. 1131-1144. | Show Abstract | Read more

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition.

Wooldridge L, Lissina A, Vernazza J, Gostick E, Laugel B, Hutchinson SL, Mirza F, Dunbar PR, Boulter JM, Glick M et al. 2007. Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region. Eur J Immunol, 37 (5), pp. 1323-1333. | Show Abstract | Read more

CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the alpha2 domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by approximately 50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 zeta chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens.

Speak AO, Salio M, Neville DC, Fontaine J, Priestman DA, Platt N, Heare T, Butters TD, Dwek RA, Trottein F et al. 2007. Implications for invariant natural killer T cell ligands due to the restricted presence of isoglobotrihexosylceramide in mammals. Proc Natl Acad Sci U S A, 104 (14), pp. 5971-5976. | Show Abstract | Read more

Development of invariant natural killer T (iNKT) cells requires the presentation of lipid ligand(s) by CD1d molecules in the thymus. The glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb3) has been proposed as the natural iNKT cell-selecting ligand in the thymus and to be involved in peripheral activation of iNKT cells by dendritic cells (DCs). However, there is no direct biochemical evidence for the presence of iGb3 in mouse or human thymus or DCs. Using a highly sensitive HPLC assay, the only tissue where iGb3 could be detected in mouse was the dorsal root ganglion (DRG). iGb3 was not detected in other mouse or any human tissues analyzed, including thymus and DCs. Even in mutant mice that store isoglobo-series GSLs in the DRG, we were still unable to detect these GSLs in the thymus. iGb3 is therefore unlikely to be a physiologically relevant iNKT cell-selecting ligand in mouse and humans. A detailed study is now warranted to better understand the nature of iNKT cell-selecting ligand(s) in vivo.

Porubsky S, Speak AO, Luckow B, Cerundolo V, Platt FM, Gröne HJ. 2007. Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency. Proc Natl Acad Sci U S A, 104 (14), pp. 5977-5982. | Show Abstract | Read more

CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Valpha14-Jalpha18 gene segments in mice and Valpha24-Jalpha18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4(+)/CD8(+) cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as alpha1-3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S(-/-)), as compared with iGb3S(+/-) mice, was confirmed. iGb3S(-/-) mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vbeta chains identical to controls. Upon administration of alpha-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S(-/-) and iGb3S(+/-) mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-gamma in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-gamma, TNF-alpha, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.

Hermans IF, Silk JD, Gileadi U, Masri SH, Shepherd D, Farrand KJ, Salio M, Cerundolo V. 2007. Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells. J Immunol, 178 (5), pp. 2721-2729. | Show Abstract

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

Ling KL, Dulphy N, Bahl P, Salio M, Maskell K, Piris J, Warren BF, George BD, Mortensen NJ, Cerundolo V. 2007. Modulation of CD103 expression on human colon carcinoma-specific CTL. J Immunol, 178 (5), pp. 2908-2915. | Show Abstract

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103alpha(E)/beta(7) by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta(7) by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta(7) in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8(+) T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta(7) in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta(7) and controlling retention of human memory CD8(+) T cells into tumor epithelium.

Chiu C, Heaps AG, Cerundolo V, McMichael AJ, Bangham CR, Callan MF. 2007. Early acquisition of cytolytic function and transcriptional changes in a primary CD8+ T-cell response in vivo. Blood, 109 (3), pp. 1086-1094. | Show Abstract | Read more

Functional studies show that programming of CD8+ T cells occurs early after initial antigen encounter within as little as 2 hours. To define the molecular basis of these events, we transferred TCR transgenic T cells from F5 Rag-/- mice into naive recipients and stimulated them with recombinant vaccinia expressing the immunodominant influenza epitope NP366-374. Transcription in epitope-specific cytotoxic T lymphocytes (CTLs) was analyzed using Affymetrix 430 2.0 GeneChips and quantitative polymerase chain reaction (PCR). We demonstrated an early transcriptional burst with the greatest number of genes reaching peak expression 12 hours after stimulation. Using in vivo cytotoxicity assays we demonstrated that early up-regulation of cytolytic genes was accompanied by acquisition of killing capacity within 24 hours of stimulation. However, T-cell proliferation was not observed until 48 hours. We therefore conclude that clonal expansion rather than acquisition of effector function is the rate-limiting step in the development of a primary CTL response.

Cerundolo V, Salio M. 2007. Harnessing NKT cells for therapeutic applications. Curr Top Microbiol Immunol, 314 pp. 325-340. | Show Abstract | Read more

Activation of NKT cells leads to the maturation of dendritic cells and efficiently assists priming of antigen-specific immune responses. The lack of polymorphism of CDld molecules and the evolutionary conservation of NKT cell responses highlight the important role of these cells in bridging innate and adaptive immune responses and advocate the value of harnessing this system in clinical settings. Compounds capable of fine tuning NKT cell activation should be actively exploited as potent adjuvants in vaccination strategies or as immunomodulators of autoimmune diseases.

Khoon LL, Pratap SE, Bates GJ, Singh B, Mortensen NJ, George BD, Warren BF, Piris J, Roncador G, Fox SB et al. 2007. Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients Cancer Immunity, 7 | Show Abstract

Recent results have shown a correlation between survival and frequency of tumour infiltrating T lymphocytes in colorectal cancer patients. However, it remains unclear whether the frequency of regulatory T cells is higher in colorectal cancer as compared to normal colon. To address this question we analysed the frequency and function of regulatory T cells in the peripheral blood and tumour infiltrating lymphocytes of colorectal cancer patients. The proportion of regulatory T cells in the peripheral blood of colorectal cancer patients (mean 8%) was significantly higher than that in normal controls (mean 2.2%). There were significantly more regulatory T cells in tumour infiltrating lymphocytes (mean 19.2%) compared to lymphocytes from an autologous non-malignant portion of the colon (mean 9%). Regulatory T cells from colorectal cancer patients were FOXP3 positive and suppressed the proliferation of autologous CD4+ CD25- cells. A higher density of tumour infiltrating regulatory T cells was found in patients with advanced as compared to early disease. These results support the hypothesis that increased numbers of regulatory T cells in the blood and tumours of colorectal cancer patients may influence the immune response against cancer and suggest that strategies to overcome regulatory T cell activity may be beneficial in the treatment of human colorectal cancer. Copyright © 2007 by Vincenzo Cerundolo.

Ling KL, Pratap SE, Bates GJ, Singh B, Mortensen NJ, George BD, Warren BF, Piris J, Roncador G, Fox SB et al. 2007. Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun, 7 pp. 7. | Show Abstract

Recent results have shown a correlation between survival and frequency of tumour infiltrating T lymphocytes in colorectal cancer patients. However, it remains unclear whether the frequency of regulatory T cells is higher in colorectal cancer as compared to normal colon. To address this question we analysed the frequency and function of regulatory T cells in the peripheral blood and tumour infiltrating lymphocytes of colorectal cancer patients. The proportion of regulatory T cells in the peripheral blood of colorectal cancer patients (mean 8%) was significantly higher than that in normal controls (mean 2.2%). There were significantly more regulatory T cells in tumour infiltrating lymphocytes (mean 19.2%) compared to lymphocytes from an autologous non-malignant portion of the colon (mean 9%). Regulatory T cells from colorectal cancer patients were FOXP3 positive and suppressed the proliferation of autologous CD4+ CD25- cells. A higher density of tumour infiltrating regulatory T cells was found in patients with advanced as compared to early disease. These results support the hypothesis that increased numbers of regulatory T cells in the blood and tumours of colorectal cancer patients may influence the immune response against cancer and suggest that strategies to overcome regulatory T cell activity may be beneficial in the treatment of human colorectal cancer.

Cerundolo V, de la Salle H. 2006. Description of HLA class I- and CD8-deficient patients: Insights into the function of cytotoxic T lymphocytes and NK cells in host defense. Semin Immunol, 18 (6), pp. 330-336. | Show Abstract | Read more

Over the last few years, several patients with defects in the HLA class I presentation pathway have been described. Analysis of their clinical symptoms and immunological parameters have led to the identification of several unexpected findings which are of importance to understand the role of HLA class I-dependent immune responses in host defense. Here, we will describe and compare clinical manifestations and immunological findings of patients with defects in the peptide transporter proteins (TAP complex), tapasin and CD8 molecules.

Mosley AJ, Meekings KN, McCarthy C, Shepherd D, Cerundolo V, Mazitschek R, Tanaka Y, Taylor GP, Bangham CR. 2006. Histone deacetylase inhibitors increase virus gene expression but decrease CD8+ cell antiviral function in HTLV-1 infection. Blood, 108 (12), pp. 3801-3807. | Show Abstract | Read more

The dynamics of human T-lymphotropic virus type-1 (HTLV-1) provirus expression in vivo are unknown. There is much evidence to suggest that HTLV-1 gene expression is restricted: this restricted gene expression may contribute to HTLV-1 persistence by limiting the ability of the HTLV-1-specific CD8(+) cell immune response to clear infected cells. In this study, we tested the hypothesis that derepression of HTLV-1 gene expression would allow an increase in CD8(+) cell-mediated lysis of HTLV-1-infected cells. Using histone deacetylase enzyme inhibitors (HDIs) to hyperacetylate histones and increase HTLV-1 gene expression, we found that HDIs doubled Tax expression in naturally infected lymphocytes after overnight culture. However, the rate of CD8(+) cell-mediated lysis of Tax-expressing cells ex vivo was halved. HDIs appeared to inhibit the CD8(+) cell-mediated lytic process itself, indicating a role for the microtubule-associated HDAC6 enzyme. These observations indicate that HDIs may reduce the efficiency of cytotoxic T-cell (CTL) surveillance of HTLV-1 in vivo. The impact of HDIs on HTLV-1 proviral load in vivo cannot be accurately predicted because of the widespread effects of these drugs on cellular processes; we therefore recommend caution in the use of HDIs in nonmalignant cases of HTLV-1 infection.

Gadola SD, Silk JD, Jeans A, Illarionov PA, Salio M, Besra GS, Dwek R, Butters TD, Platt FM, Cerundolo V. 2006. Impaired selection of invariant natural killer T cells in diverse mouse models of glycosphingolipid lysosomal storage diseases. J Exp Med, 203 (10), pp. 2293-2303. | Show Abstract | Read more

Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to alpha-galactosylceramide (alpha-GalCer) and reduced iNKT cell-mediated lysis of wild-type targets loaded with alpha-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4(+) T cell responses were generally unaffected, processing of a lysosome-dependent analogue of alpha-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands.

Palmowski MJ, Gileadi U, Salio M, Gallimore A, Millrain M, James E, Addey C, Scott D, Dyson J, Simpson E, Cerundolo V. 2006. Role of immunoproteasomes in cross-presentation. J Immunol, 177 (2), pp. 983-990. | Show Abstract

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty(246-254) epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7(-/-)) skin grafts by female LMP7(-/-) mice. Second, we ruled out an altered Uty(246-254)-specific T cell repertoire in LMP7(-/-) female mice and demonstrated efficient Uty(246-254) presentation by re-expressing LMP7 in male LMP7(-/-) cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty(246-254)-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7(-/-) female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.

Purbhoo MA, Sutton DH, Brewer JE, Mullings RE, Hill ME, Mahon TM, Karbach J, Jäger E, Cameron BJ, Lissin N et al. 2006. Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors. J Immunol, 176 (12), pp. 7308-7316. | Show Abstract

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.

Lau YS, Sabokbar A, Giele H, Cerundolo V, Hofstetter W, Athanasou NA. 2006. Malignant melanoma and bone resorption. Br J Cancer, 94 (10), pp. 1496-1503. | Show Abstract | Read more

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Zhang J, Raper A, Sugita N, Hingorani R, Salio M, Palmowski MJ, Cerundolo V, Crocker PR. 2006. Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors. Blood, 107 (9), pp. 3600-3608. | Show Abstract | Read more

We describe the cloning and characterization of Siglec-H, a novel murine CD33-related siglec-like molecule with 2 immunoglobulin domains. Unlike other CD33-related siglecs, Siglec-H lacks tyrosine-based signaling motifs in its cytoplasmic tail. Although Siglec-H has the typical structural features required for sialic acid binding, no evidence for carbohydrate recognition was obtained. Specific monoclonal and polyclonal antibodies (Abs) were raised to Siglec-H and used to define its cellular expression pattern and functional properties. By flow cytometry, Siglec-H was expressed specifically on plasmacytoid dendritic cell (pDC) precursors in bone marrow, spleen, blood, and lymph nodes. Staining of tissue sections showed that Siglec-H was also expressed in a subset of marginal zone macrophages in the spleen and in medullary macrophages in lymph nodes. Using bone marrow-derived pDC precursors that express Siglec-H, addition of Abs did not influence cytokine production, either in the presence or absence of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG). In comparison, Siglec-H functioned as an endocytic receptor and mediated efficient internalization of anti-Siglec-H Abs. By immunizing mice with ovalbumin-conjugated anti-Siglec-H Ab in the presence of CpG, we demonstrate generation of antigen-specific CD8 T cells in vivo. Targeting Siglec-H may therefore be a useful way of delivering antigens to pDC precursors for cross-presentation.

Quiroga I, Salio M, Koo DD, Cerundolo L, Shepherd D, Cerundolo V, Fuggle SV. 2006. Expression of MHC class I-related Chain B (MICB) molecules on renal transplant biopsies. Transplantation, 81 (8), pp. 1196-1203. | Show Abstract | Read more

BACKGROUND: MICA and MICB (MHC class I-related chain A and B) are polymorphic genes that encode molecules related to MHC class I and are expressed on epithelial cells in response to stress. Incompatible donor MIC antigens can stimulate antibody production in transplant recipients. This study was designed to determine MICB expression in kidney pretransplant and any subsequent changes in expression following transplantation and to correlate changes with inflammatory markers and clinical events. METHODS: Paired renal biopsies obtained from living donor (n=10) and cadaveric allografts (n=50) before and 7 days posttransplant were stained for MICB, leukocytic infiltration, and HLA class II antigens. RESULTS: Variable tubular MICB expression was evident in donor biopsies [high 6/60 (10%), low/negative 13/60 (22%), intermediate 41/60 (68%)]. Following transplantation, MICB was up-regulated on renal tubules of 17/60 (28%) biopsies and was associated with MHC class II antigen induction (P=0.02) and leukocyte infiltration (P=0.01). Acute tubular necrosis leading to delayed graft function (DGF) and acute rejection (AR) cause cellular stress within the transplanted kidney. We found a strong association between up-regulation of MICB and cellular stress, 15/17 biopsies with up-regulated MICB expression had AR and/or DGF (P=0.003). CONCLUSIONS: This is the first study demonstrating variable levels of MICB expression in kidneys before transplantation and induction of MICB expression following renal transplantation. MICB expression is associated with HLA class II antigen induction, leukocytic infiltration of the graft and cellular stress in the transplanted kidney. Expression of MICB could contribute significantly to the alloimmune response in mismatched donors and recipients.

Kotsianidis I, Silk JD, Spanoudakis E, Patterson S, Almeida A, Schmidt RR, Tsatalas C, Bourikas G, Cerundolo V, Roberts IA, Karadimitris A. 2006. Regulation of hematopoiesis in vitro and in vivo by invariant NKT cells. Blood, 107 (8), pp. 3138-3144. | Show Abstract | Read more

Invariant natural killer T cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. On activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete T helper 1 and 2 (Th1/2) cytokines but also cytokines with hematopoietic potential such as GM-CSF. Here, we show that the myeloid clonogenic potential of human hematopoietic progenitors is increased in the presence of glycolipid-activated, GM-CSF-secreting NKT cells; conversely, short- and long-term progenitor activity is decreased in the absence of NKT cells, implying regulation of hematopoiesis in both the presence and the absence of immune activation. In accordance with these findings, iNKT-cell-deficient mice display impaired hematopoiesis characterized by peripheral-blood cytopenias, reduced marrow cellularity, lower frequency of hematopoietic stem cells (HSCs), and reduced early and late hematopoietic progenitors. We also show that CD1d is expressed on human HSCs. CD1d-expressing HSCs display short- and long-term clonogenic potential and can present the glycolipid alpha-galactosylceramide to iNKT cells. Thus, iNKT cells emerge as the first subset of regulatory T cells that are required for effective hematopoiesis in both steady-state conditions and under conditions of immune activation.

Gadola SD, Koch M, Marles-Wright J, Lissin NM, Shepherd D, Matulis G, Harlos K, Villiger PM, Stuart DI, Jakobsen BK et al. 2006. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors. J Exp Med, 203 (3), pp. 699-710. | Show Abstract | Read more

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Ait-Tahar K, Cerundolo V, Banham AH, Hatton C, Blanchard T, Kusec R, Becker M, Smith GL, Pulford K. 2006. B and CTL responses to the ALK protein in patients with ALK-positive ALCL. Int J Cancer, 118 (3), pp. 688-695. | Show Abstract | Read more

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) has a good prognosis compared to ALK-negative ALCL, possibly as a result of the immune recognition of the ALK proteins. The aim of our study was to investigate the presence of both a B and cytotoxic T cell (CTL) response to ALK in ALK-positive ALCL. We confirmed the presence of an antibody response to ALK in all 9 ALK-positive ALCL patients investigated. An ELISpot assay was used to detect a gamma-interferon (IFN) T cell response after short term culture of mononuclear blood cells with 2 ALK-derived HLA-A*0201 restricted peptides: ALKa and ALKb. A significant gamma-IFN response was identified in all 7 HLA-A*0201-positive ALK-positive ALCL patients but not in ALK-negative ALCL patients (n = 2) or normal subjects (n = 6). CTL lines (>95% CD8-positive) raised from 2 ALK-positive ALCL patients lysed ALK-positive ALCL derived cell lines in a MHC-Class I restricted manner. This is the first report of both a B cell and CTL response to ALK in patients with ALK-positive ALCL. This response persisted during long-term remission. The use of modified vaccinia virus Ankara (MVA) to express ALK is also described. Our findings are of potential prognostic value and open up therapeutic options for those ALK-positive patients who do not respond to conventional treatment.

Chen N, McCarthy C, Drakesmith H, Li D, Cerundolo V, McMichael AJ, Screaton GR, Xu XN. 2006. HIV-1 down-regulates the expression of CD1d via Nef. Eur J Immunol, 36 (2), pp. 278-286. | Show Abstract | Read more

HIV-1 has evolved several strategies to subvert host immune responses to the infected cells. One is to inhibit CTL recognition by HIV-1 Nef-mediated down-regulation of MHC-I expression on the surface of infected cells. Here we report that Nef also reduces the expression of the non-classical MHC-I like CD1d molecule, a third lineage of antigen-presenting molecule, which presents lipid antigens. Nef achieves this by increasing internalization of CD1d molecules from the cell surface and retaining CD1d in the trans-Golgi-network (TGN). This effect depends on a tyrosine-based motif present in CD1 cytoplasmic tail as well as the actions of four Nef motifs, which are known to be involved in the down-regulation of MHC-I or CD4. These results suggest that Nef regulates intracellular trafficking of CD1d via a distinct but shared pathway with MHC-I and CD4. Thus, HIV-1 reduces the visibility of its infected cells not only to MHC-I-restricted T cells but also to CD1d-restricted NKT cells. Given that CD1d-restricted T cells have unique effector and regulatory functions in innate and adapted immune responses as compared with their counterpart MHC-restricted T cells, our data provide additional new insights into molecular basis of HIV-1-mediated damage to the immune system.

Pasquetto V, Bui HH, Giannino R, Mirza F, Sidney J, Oseroff C, Tscharke DC, Irvine K, Bennink JR, Peters B et al. 2005. HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products (vol 175, pg 5504, 2005) JOURNAL OF IMMUNOLOGY, 175 (12), pp. 8440-8440.

Smith CL, Mirza F, Pasquetto V, Tscharke DC, Palmowski MJ, Dunbar PR, Sette A, Harris AL, Cerundolo V. 2005. Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara. J Immunol, 175 (12), pp. 8431-8437. | Show Abstract

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.

Silk JD, Gadola SD, Jeans A, Illarionov PA, Besra G, Dwek R, Butters TD, Platt FM, Cerundolo V. 2005. Impaired selection of invariant natural killer T cells in diverse mouse models of glycosphingolipid lysosomal storage diseases IMMUNOLOGY, 116 pp. 66-67.

Trautmann L, Rimbert M, Echasserieau K, Saulquin X, Neveu B, Dechanet J, Cerundolo V, Bonneville M. 2005. Selection of T cell clones expressing high-affinity public TCRs within Human cytomegalovirus-specific CD8 T cell responses. J Immunol, 175 (9), pp. 6123-6132. | Show Abstract

Assessment of clonal diversity of T cell responses against human CMV (HCMV), a major cause of morbidity in immunodepressed patients, provides important insights into the molecular basis of T cell immunodominance, and has also clinical implications for the immunomonitoring and immunotherapy of HCMV infections. We performed an in-depth molecular and functional characterization of CD8 T cells directed against an immunodominant HLA-A2-restricted epitope derived from HCMV protein pp65 (NLV/A2) in steady state and pathological situations associated with HCMV reactivation. NLV/A2-specific T cells in healthy HCMV-seropositive donors showed limited clonal diversity and usage of a restricted set of TCR Vbeta regions. Although TCRbeta-chain junctional sequences were highly diverse, a large fraction of NLV/A2-specific T cells derived from distinct individuals showed several recurrent (so-called "public") TCR features associated in some cases with full conservation of the TCRalpha chain junctional region. A dramatic clonal focusing of NLV/A2-specific T cells was observed in situations of HCMV reactivation and/or chronic inflammation, which resulted in selection of a single clonotype displaying similar public TCR features in several patients. In most instances the NLV/A2-specific dominant clonotypes showed higher affinity for their Ag than subdominant ones, thus suggesting that TCR affinity/avidity is the primary driving force underlying repertoire focusing along chronic antigenic stimulation.

Pasquetto V, Bui HH, Giannino R, Banh C, Mirza F, Sidney J, Oseroff C, Tscharke DC, Irvine K, Bennink JR et al. 2005. HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products. J Immunol, 175 (8), pp. 5504-5515. | Show Abstract

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome approximately 20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8(+) T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.

Patterson S, Kotsianidis I, Almeida A, Politou M, Rahemtulla A, Matthew B, Schmidt RR, Cerundolo V, Roberts IA, Karadimitris A. 2005. Human invariant NKT cells are required for effective in vitro alloresponses. J Immunol, 175 (8), pp. 5087-5094. | Show Abstract

NKT cells are a small subset of regulatory T cells conserved in humans and mice. In humans they express the Valpha24Jalpha18 invariant chain (hence invariant NKT (iNKT) cells) and are restricted by the glycolipid-presenting molecule CD1d. In mice, iNKT cells may enhance or inhibit anti-infectious and antitumor T cell responses but suppress autoimmune and alloreactive responses. We postulated that iNKT cells might also modulate human alloreactive responses. Using MLR assays we demonstrate that in the presence of the CD1d-presented glycolipid alpha-galactosylceramide (alphaGC) alloreactivity is enhanced (37 +/- 12%; p < 0.001) in an iNKT cell-dependent manner. iNKT cells are activated early during the course of the MLR, presumably by natural ligands. In MLR performed without exogenous ligands, depletion of iNKT cells significantly diminished the alloresponse in terms of proliferation (58.8 +/- 24%; p < 0.001) and IFN-gamma secretion (43.2 +/- 15.2%; p < 0.001). Importantly, adding back fresh iNKT cells restored the reactivity of iNKT cell-depleted MLR to near baseline levels. CD1d-blocking mAbs equally reduced the reactivity of the iNKT cell-replete and -depleted MLR compared with IgG control, indicating that the effect of iNKT cells in the in vitro alloresponse is CD1d-dependent. These findings suggest that human iNKT cells, although not essential for its development, can enhance the alloreactive response.

Milicic A, Price DA, Zimbwa P, Booth BL, Brown HL, Easterbrook PJ, Olsen K, Robinson N, Gileadi U, Sewell AK et al. 2005. CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef. J Immunol, 175 (7), pp. 4618-4626. | Show Abstract

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.

Koch M, Stronge VS, Shepherd D, Gadola SD, Mathew B, Ritter G, Fersht AR, Besra GS, Schmidt RR, Jones EY, Cerundolo V. 2005. The crystal structure of human CD1d with and without alpha-galactosylceramide. Nat Immunol, 6 (8), pp. 819-826. | Show Abstract | Read more

The glycolipid alpha-galactosylceramide binds with high affinity to CD1d and stimulates natural killer T cells. Here we report the crystal structure of human CD1d in complex with synthetic alpha-galactosylceramide at a resolution of 3.0 A. The structure shows a tightly fit lipid in the CD1d binding groove, with the sphingosine chain bound in the C' pocket and the longer acyl chain anchored in the A' pocket. We also present the CD1d structure without lipid, which has a more open conformation of the binding groove, suggesting a dual conformation of CD1d in which the 'open' conformation is more able to load lipids. These structures provide clues as to how CD1 molecules load glycolipids as well as data to guide the design of new therapeutic agents.

Maczek C, Berger TG, Schuler-Thurner B, Schultz ES, Hamann A, Dunbar PR, Cerundolo V, Steinkasserer A, Schuler G. 2005. Differences in phenotype and function between spontaneously occurring melan-A-, tyrosinase- and influenza matrix peptide-specific CTL in HLA-A*0201 melanoma patients. Int J Cancer, 115 (3), pp. 450-455. | Show Abstract | Read more

Melanoma-specific T cells can occur spontaneously or in response to vaccination or other therapies, but the frequency is much lower than observed in viral infections. The presence of tumor-specific T cells does not necessarily translate into clinical regressions for a variety of reasons such as an insufficient frequency, activation state or homing capacity of the T cells or escape strategies of the tumor. Having screened melanoma patients prior to inclusion in vaccination trials for spontaneous tumor-specific T cells either by Elispot or tetramer-staining, we have identified 3 patients with sufficient numbers of tumor-reactive T cells to more than 1 TAA and at least 1 virus-antigen to perform phenotypic and functional analysis directly ex vivo. These stage IV melanoma patients showed specific CTL against melan-A.A2, tyrosinase.A2 and influenza matrix peptide (IMP).A2 readily detectable in peripheral blood. T-cell receptor (TCR) staining using the tetramer technology was combined with phenotypic characterization and functional assays. In contrast to IMP-specific CTL, melanoma-specific CTL were predominantly terminally differentiated effector cells. However, analysis of melan-A- and tyrosinase-specific T-cell lines showed that only a part of the melanoma-specific CTL were able to lyse peptide-loaded target cells. Interestingly, the described phenotypic and functional differences of melan-A- and tyrosinase-specific CTL appeared not only between patients but were also evident within patients, suggesting that the immune response against various tumor antigens is regulated independently.

Roncador G, Brown PJ, Maestre L, Hue S, Martínez-Torrecuadrada JL, Ling KL, Pratap S, Toms C, Fox BC, Cerundolo V et al. 2005. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol, 35 (6), pp. 1681-1691. | Show Abstract | Read more

The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

Manders PM, Hunter PJ, Telaranta AI, Carr JM, Marshall JL, Carrasco M, Murakami Y, Palmowski MJ, Cerundolo V, Kaech SM et al. 2005. BCL6b mediates the enhanced magnitude of the secondary response of memory CD8+ T lymphocytes. Proc Natl Acad Sci U S A, 102 (21), pp. 7418-7425. | Show Abstract | Read more

A characteristic of the secondary response of CD8(+) T cells that distinguishes it from the primary response is the generation of greater numbers of effector cells. Because effector CD8(+) T cells are derived from a pool of less differentiated, replicating cells in secondary lymphoid organs, and because IL-2 mediates effector differentiation, the enhanced secondary response may reflect the enlargement of this generative pool by the transient repression of IL-2-mediated differentiation. We have examined for this function the transcriptional repressor BCL6b, a homologue of BCL6 that represses IL-2-induced B cell differentiation. BCL6b is expressed in a small subset of antigen-experienced CD8(+) T cells. Ectopic expression of BCL6b in CD8(+) T cells diminishes their growth in response to IL-2 in vitro. Female mice in which the BCL6b gene has been interrupted have normal primary responses of CD8(+) T cells to infection with vaccinia expressing the H-Y epitope, Uty, but Uty-specific, BCL6b(-/-), memory CD8(+) T cells have diminished recall proliferative responses to this epitope in vitro. BCL6b(-/-) mice also have normal primary CD8(+) T cell responses to influenza infection, but nucleoprotein peptide-specific, BCL6b(-/-), memory CD8(+) T cells have a cell autonomous defect in the number of effector cells generated in response to reinfection. Therefore, BCL6b is required for the enhanced magnitude of the secondary response of memory CD8(+) T cells.

Salio M, Cerundolo V. 2005. Viral immunity: cross-priming with the help of TLR3. Curr Biol, 15 (9), pp. R336-R339. | Show Abstract | Read more

Cross-presentation is important for regulating T-cell responses to exogenous antigens and can maintain tolerance (cross-tolerance) or induce immune responses (cross-priming). Recent exciting results on the role of the Toll-like receptor TLR3 in promoting cross-priming of viral antigens provide new insights into the mechanisms that allow Toll-like receptor signaling to bridge innate and adaptive immune responses.

Chen JL, Stewart-Jones G, Bossi G, Lissin NM, Wooldridge L, Choi EM, Held G, Dunbar PR, Esnouf RM, Sami M et al. 2005. Structural and kinetic basis for heightened immunogenicity of T cell vaccines. J Exp Med, 201 (8), pp. 1243-1255. | Show Abstract | Read more

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.

Renneson J, Salio M, Mazouz N, Goldman M, Marchant A, Cerundolo V. 2005. Mature dendritic cells differentiated in the presence of interferon-beta and interleukin-3 prime functional antigen-specific CD8 T cells. Clin Exp Immunol, 139 (3), pp. 468-475. | Show Abstract | Read more

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-beta and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-beta/IL-3 DCs) that are particularly efficient at eliciting IFN-gamma and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8(+) T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-beta/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A(26-35)-specific CD8(+) T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8(+) T cells produced IFN-gamma and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8(+) T cell numbers was generally higher following priming with CD40-L than with polyinosinic-polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-beta/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.

Smith CL, Dunbar PR, Mirza F, Palmowski MJ, Shepherd D, Gilbert SC, Coulie P, Schneider J, Hoffman E, Hawkins R et al. 2005. Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence. Int J Cancer, 113 (2), pp. 259-266. | Show Abstract | Read more

Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a "prime-boost" DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A(26-35) analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naive patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029-0.19% CD8(+) T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.

Pasquetto V, Bui HH, Giannino R, Banh C, Mirza F, Sidney J, Oseroff C, Tscharke DC, Irvine K, Bennink JR et al. 2005. Erratum: HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products (Journal of Immunology (2005) 175 (5504-5515)) Journal of Immunology, 175 (12), pp. 8440.

Collins MK, Cerundolo V. 2004. Gene therapy meets vaccine development. Trends Biotechnol, 22 (12), pp. 623-626. | Show Abstract | Read more

Therapeutic vaccines such as those used to combat cancer or persistent viral infection are required to reprogramme a downregulated immune system. This presents a difficult challenge for vaccine design and merits the development of novel immunization protocols. Currently, we know that mobilization of dendritic cells (DCs) to present antigens to T lymphocytes is crucial for effective immunization. Our increasing understanding of DC biology, coupled with the growing sophistication of viral vectors developed for gene therapy, makes more rational vaccine design an exciting possibility. Here we propose that engineering viral vectors to express antigens in activated DCs will provide the most effective vaccines for priming an immune response.

Silk JD, Hermans IF, Gileadi U, Chong TW, Shepherd D, Salio M, Mathew B, Schmidt RR, Lunt SJ, Williams KJ et al. 2004. Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell-mediated immunotherapy. J Clin Invest, 114 (12), pp. 1800-1811. | Show Abstract | Read more

Activation of invariant CD1d-dependent NK T cells (iNKT cells) in vivo through administration of the glycolipid ligand alpha-galactosylceramide (alpha-GalCer) or the sphingosine-truncated alpha-GalCer analog OCH leads to CD40 signaling as well as the release of soluble molecules including type 1 and gamma interferons that contribute to DC maturation. This process enhances T cell immunity to antigens presented by the DC. The adjuvant activity is further amplified if APCs are stimulated through Toll-like receptor 4, suggesting that iNKT cell signals can amplify maturation induced by microbial stimuli. The adjuvant activity of alpha-GalCer enhances both priming and boosting of CD8(+) T cells to coadministered peptide or protein antigens, including a peptide encoding the clinically relevant, HLA-A2-restricted epitope of the human tumor antigen NY-ESO-1. Importantly, alpha-GalCer was used to induce CD8(+) T cells to antigens delivered orally, despite the fact that this route of administration is normally associated with blunted responses. Only T cell responses induced in the presence of iNKT cell stimulation, whether by the i.v. or oral route, were capable of eradicating established tumors. Together these data highlight the therapeutic potential of iNKT cell ligands in vaccination strategies, particularly "heterologous prime-boost" strategies against tumors, and provide evidence that iNKT cell stimulation may be exploited in the development of oral vaccines.

Barnes E, Salio M, Cerundolo V, Medlin J, Murphy S, Dusheiko G, Klenerman P. 2004. Impact of alpha interferon and ribavirin on the function of maturing dendritic cells. Antimicrob Agents Chemother, 48 (9), pp. 3382-3389. | Show Abstract | Read more

Alpha interferon and ribavirin are required in combination to achieve a sustained virological response in the treatment of hepatitis C virus (HCV) infection. Alpha interferon has direct antiviral activity and also enhances HCV-specific T-cell responses. Ribavirin has little direct activity against HCV but reduces hepatic inflammation. It is therefore likely that these drugs in combination have hitherto unidentified immunological effects. In the present study we investigated the effects of alpha interferon and ribavirin on dendritic cell (DC) maturation and cytokine production induced by double-stranded RNA in vitro. Alpha interferon alone enhanced the expression of HLA class I, HLA class II, and CD86 on immature DCs but did not stimulate full DC maturation, which requires the expression of CD83. Alpha interferon enhanced the production of interleukin 12 p70 [IL-12(p70)] and tumor necrosis factor alpha (TNF-alpha) but had no effect on IL-10 production. In contrast, ribavirin at physiological doses had no effect on DC maturation but markedly suppressed the production of TNF-alpha, IL-10, and IL-12(p70). The suppression of cytokines by ribavirin cannot be explained by the induction of DC apoptosis or cell death. Quantitative PCR confirmed that cytokine suppression occurs at the level of mRNA. The suppression of IL-12(p70) and TNF-alpha in maturing DCs may explain the reduction in hepatic inflammation observed during ribavirin monotherapy. Combination alpha interferon-ribavirin therapy may alter the cytokine profile of maturing DCs overall by suppressing IL-10 production but maintaining IL-12(p70) and TNF-alpha production, a pattern that would favor viral elimination through downstream effects on T cells.

Markel G, Achdout H, Katz G, Ling KL, Salio M, Gruda R, Gazit R, Mizrahi S, Hanna J, Gonen-Gross T et al. 2004. Biological function of the soluble CEACAM1 protein and implications in TAP2-deficient patients. Eur J Immunol, 34 (8), pp. 2138-2148. | Show Abstract | Read more

Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory signals controlling NK killing activity. It is therefore surprising to learn that TAP2-deficient patients suffer from autoimmune manifestations only occasionally in later stages of life. We have previously described that the CEACAM1-mediated inhibitory mechanism of NK cytotoxicity plays a major role in controlling NK autoreactivity in three newly identified TAP2-deficient siblings. This novel mechanism probably compensates for the lack of MHC class I-mediated inhibition. The CEACAM1 protein can also be present in a soluble form and the biological function of the soluble form of CEACAM1 with regard to NK cells has not been investigated. Here we show that the homophilic CEACAM1 interactions are abrogated in the presence of soluble CEACAM1 protein in a dose-dependent manner. Importantly, the amounts of soluble CEACAM1 protein detected in sera derived from the TAP2-deficient patients were dramatically reduced as compared to healthy controls. This dramatic reduction does not depend on the membrane-bound metalloproteinase activity. Thus, the expression of CEACAM1 and the absence of soluble CEACAM1 observed in the TAP2-deficient patients practically maximize the inhibitory effect and probably help to minimize autoimmunity in these patients.

Smith DC, Salio M, Lord JM, Roberts LM, Cerundolo V. 2004. Lack of dendritic cell maturation by the plant toxin ricin. Eur J Immunol, 34 (8), pp. 2149-2157. | Show Abstract | Read more

Several bacterial toxins either promote or inhibit the maturation of human monocyte-derived DC. Since the potent plant toxin ricin exploits the same cell entry pathway used by these bacterial toxins and shares identical catalytic activity with some of them, we have studied the capacity of ricin to induce DC maturation in vitro. Here, we show that in contrast to the bacterial proteins, ricin neither induces DC maturation nor interferes with LPS-induced DC maturation. There is no correlation between the absence of DC maturation and ricin dysfunction. Indeed, some of the ricin variants retain significant ribotoxicity and catalytic activity. We have extended these observations to ebulin-1, suggesting that this may be a general characteristic of plant-derived cytotoxic ribosome-inactivating toxins. The human immune system may therefore have evolved to recognize and rapidly respond to the bacterial proteins, whilst being less responsive to the equivalent plant cytotoxins. Understanding the effect of ricin on professional APC may provide insights into the generation of an anti-ricin vaccine and into the use of inactivated ricin A chains as delivery vectors as part of a vaccination protocol.

Markel G, Mussaffi H, Ling KL, Salio M, Gadola S, Steuer G, Blau H, Achdout H, de Miguel M, Gonen-Gross T et al. 2004. The mechanisms controlling NK cell autoreactivity in TAP2-deficient patients. Blood, 103 (5), pp. 1770-1778. | Show Abstract | Read more

The killing of natural killer (NK) cells is regulated by activating and inhibitory NK receptors that recognize mainly class I major histocompatibility complex (MHC) proteins. In transporter associated with antigen processing (TAP2)-deficient patients, killing of autologous cells by NK cells is therefore expected. However, none of the TAP2-deficient patients studied so far have suffered from immediate NK-mediated autoimmune manifestations. We have previously demonstrated the existence of a novel class I MHC-independent inhibitory mechanism of NK cell cytotoxicity mediated by the homophilic carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) interactions. Here, we identified 3 new siblings suffering from TAP2 deficiency. NK cells derived from these patients express unusually high levels of the various killer cell inhibitory receptors (KIRs) and the CEACAM1 protein. Importantly, the patients' NK cells use the CEACAM1 protein to inhibit the killing of tumor and autologous cells. Finally, we show that the function of the main NK lysis receptor, NKp46, is impaired in these patients. These results indicate that NK cells in TAP2-deficient patients have developed unique mechanisms to reduce NK killing activity and to compensate for the lack of class I MHC-mediated inhibition. These mechanisms prevent the attack of self-cells by the autologous NK cells and explain why TAP2-deficient patients do not suffer from autoimmune manifestations in early stages of life.

Salio M, Palmowski MJ, Atzberger A, Hermans IF, Cerundolo V. 2004. CpG-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional CD8 T cell responses to endogenous but not exogenous antigens. J Exp Med, 199 (4), pp. 567-579. | Show Abstract | Read more

Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor-deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-gamma and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

Batuwangala T, Shepherd D, Gadola SD, Gibson KJ, Zaccai NR, Fersht AR, Besra GS, Cerundolo V, Jones EY. 2004. The crystal structure of human CD1b with a bound bacterial glycolipid. J Immunol, 172 (4), pp. 2382-2388. | Show Abstract

The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains. A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons. The structure of CD1b complexed with glucose monomycolate, reported in this study, confirms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be accommodated. The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact accommodation of lengthy acyl chains. The current crystal structure illustrates for the first time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features fit this molecule for its role in the immune response to intracellular bacteria.

Papagno L, Spina CA, Marchant A, Salio M, Rufer N, Little S, Dong T, Chesney G, Waters A, Easterbrook P et al. 2004. Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection. PLoS Biol, 2 (2), pp. E20. | Show Abstract | Read more

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8(+) T-cells and the use of an in vitro model of naïve CD8(+) T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8(+) T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8(+) T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8(+) and CD4(+) T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.

Palmowski MJ, Lopes L, Ikeda Y, Salio M, Cerundolo V, Collins MK. 2004. Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response. J Immunol, 172 (3), pp. 1582-1587. | Show Abstract

Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1(157-165)-specific CD8(+) cells, detected ex vivo with an A2/H-2K(b) chimeric class I tetramer. These NY-ESO-1(157-165)-specific CD8(+) cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1(157-165)-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.

Hermans IF, Silk JD, Yang J, Palmowski MJ, Gileadi U, McCarthy C, Salio M, Ronchese F, Cerundolo V. 2004. The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo. J Immunol Methods, 285 (1), pp. 25-40. | Show Abstract | Read more

Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.

Cerundolo V, Hermans IF, Salio M. 2004. Dendritic cells: a journey from laboratory to clinic. Nat Immunol, 5 (1), pp. 7-10. | Show Abstract | Read more

Dendritic cell-based vaccines have been rapidly transferred from the laboratory to the clinic. As the full potential of these cells has not yet been entirely exploited, many strategies could improve the immunogenicity of these vaccines.

Oppenheim J, Blankenstein T, Brennan F, Gordon S, Mantovani A, Strieter RM, Cerundolo V, Forni G, Thun MJ, Balkwill F, Richmond A. 2004. Discussion Novartis Foundation Symposium, 256 pp. 210-214.

Brennan F, Mantovani A, Gordon S, Cerundolo V, Pollard JW, Forni G, Harris A, Balkwill F, Richmond A, Oppenheim J. 2004. Discussion Novartis Foundation Symposium, 256 pp. 146-148.

Pollard JW, Caux C, Strieter RM, Brennan F, Cerundolo V, Pepper MS, Gallimore A, Blankenstein T, Oppenheim J, Feldmann M et al. 2004. Discussion Novartis Foundation Symposium, 256 pp. 254-258.

Blankenstein T, Pepper MS, Harris A, Balkwill F, Cerundolo V, Gordon S, Rollins B, Richmond A, Oppenheim J, Mantovani A et al. 2004. Discussion Novartis Foundation Symposium, 256 pp. 132-136.

Wooldridge L, Hutchinson SL, Choi EM, Lissina A, Jones E, Mirza F, Dunbar PR, Price DA, Cerundolo V, Sewell AK. 2003. Anti-CD8 antibodies can inhibit or enhance peptide-MHC class I (pMHCI) multimer binding: this is paralleled by their effects on CTL activation and occurs in the absence of an interaction between pMHCI and CD8 on the cell surface. J Immunol, 171 (12), pp. 6650-6660. | Show Abstract

Cytotoxic T lymphocytes recognize short peptides presented in association with MHC class I (MHCI) molecules on the surface of target cells. The Ag specificity of T lymphocytes is conferred by the TCR, but invariable regions of the peptide-MHCI (pMHCI) molecule also interact with the cell surface glycoprotein CD8. The distinct binding sites for CD8 and the TCR allow pMHCI to be bound simultaneously by both molecules. Even before it was established that the TCR recognized pMHCI, it was shown that CTL exhibit clonal heterogeneity in their ability to activate in the presence of anti-CD8 Abs. These Ab-based studies have since been interpreted in the context of the interaction between pMHCI and CD8 and have recently been extended to show that anti-CD8 Ab can affect the cell surface binding of multimerized pMHCI Ags. In this study, we examine the role of CD8 further using point-mutated pMHCI Ag and show that anti-CD8 Abs can either enhance or inhibit the activation of CTL and the stable cell surface binding of multimerized pMHCI, regardless of whether there is a pMHCI/CD8 interaction. We further demonstrate that multimerized pMHCI Ag can recruit CD8 in the absence of a pMHCI/CD8 interaction and that anti-CD8 Abs can generate an intracellular activation signal resulting in CTL effector function. These results question many previous assumptions as to how anti-CD8 Abs must function and indicate that CD8 has multiple roles in CTL activation that are not necessarily dependent on an interaction with pMHCI.

Hermans IF, Chong TW, Palmowski MJ, Harris AL, Cerundolo V. 2003. Synergistic effect of metronomic dosing of cyclophosphamide combined with specific antitumor immunotherapy in a murine melanoma model. Cancer Res, 63 (23), pp. 8408-8413. | Show Abstract

Immunotherapy could be combined with conventional chemotherapeutic modalities aimed at reducing tumor burden. Such combination therapy may be most useful when "metronomic" doses of antineoplastic drugs are used, thereby potentially avoiding some of the immunosuppressive effects of these drugs. Recent studies have shown that some conventional antineoplastic drugs can be exploited for antiangiogenic capacities, a strategy that requires drugs to be administered at regular intervals. We therefore investigated whether such metronomic therapy with the alkylating agent cyclophosphamide (CTX) could be effectively combined with immunotherapy eliciting tumor-reactive CTLs. An immunization protocol using injection of recombinant DNA followed by injection of recombinant modified vaccinia virus Ankara strain was used to initiate a specific CTL response in mice capable of providing resistance to challenge with the murine melanoma B16.F10. Combining this immunotherapeutic regime with metronomic delivery of CTX resulted in antitumor activity that was dramatically enhanced over either treatment administered alone and was also significantly greater than combining immunotherapy with CTX administered by a maximum tolerated dose regime. Whereas both metronomic and maximum tolerated dose delivery of CTX did cause deletion of proliferating tumor-specific CTLs in the blood, this deletion occurred with slower kinetics with the metronomic schedule. Further analysis showed that metronomic CTX treatment did not delete cells with low expression of CD43, a "memory" phenotype, and that these cells maintained potent restimulatory capacity. The combination of immunotherapy and metronomic CTX therapy may be well suited to clinical management of cancer.

Faroudi M, Utzny C, Salio M, Cerundolo V, Guiraud M, Müller S, Valitutti S. 2003. Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold. Proc Natl Acad Sci U S A, 100 (24), pp. 14145-14150. | Show Abstract | Read more

Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).

Choi EM, Chen JL, Wooldridge L, Salio M, Lissina A, Lissin N, Hermans IF, Silk JD, Mirza F, Palmowski MJ et al. 2003. High avidity antigen-specific CTL identified by CD8-independent tetramer staining. J Immunol, 171 (10), pp. 5116-5123. | Show Abstract

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.

Hermans IF, Silk JD, Gileadi U, Salio M, Mathew B, Ritter G, Schmidt R, Harris AL, Old L, Cerundolo V. 2003. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol, 171 (10), pp. 5140-5147. | Show Abstract

Modification in the function of dendritic cells (DC), such as that achieved by microbial stimuli or T cell help, plays a critical role in determining the quality and size of adaptive responses to Ag. NKT cells bearing an invariant TCR (iNKT cells) restricted by nonpolymorphic CD1d molecules may constitute a readily available source of help for DC. We therefore examined T cell responses to i.v. injection of soluble Ag in the presence or the absence of iNKT cell stimulation with the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GalCer). Considerably enhanced CD4(+) and CD8(+) T cell responses were observed when alpha-GalCer was administered at the same time as or close to OVA injection. This enhancement was dependent on the involvement of iNKT cells and CD1d molecules and required CD40 signaling. Studies in IFN-gammaR(-/-) mice indicated that IFN-gamma was not required for the adjuvant effect of alpha-GalCer. Consistent with this result, enhanced T cell responses were observed using OCH, an analog of alpha-GalCer with a truncated sphingosine chain and a reduced capacity to induce IFN-gamma. Splenic DC from alpha-GalCer-treated animals expressed high levels of costimulatory molecules, suggesting maturation in response to iNKT cell activation. Furthermore, studies with cultured DC indicated that potentiation of T cell responses required presentation of specific peptide and alpha-GalCer by the same DC, implying conditioning of DC by iNKT cells. The iNKT-enhanced T cell responses resisted challenge with OVA-expressing tumors, whereas responses induced in the absence of iNKT stimulation did not. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble Ag by modulating DC function.

Salio M, Dulphy N, Renneson J, Herbert M, McMichael A, Marchant A, Cerundolo V. 2003. Efficient priming of antigen-specific cytotoxic T lymphocytes by human cord blood dendritic cells. Int Immunol, 15 (10), pp. 1265-1273. | Show Abstract | Read more

Previous studies have suggested that defective immune responses in early life may be related to the immaturity of neonatal antigen-presenting cells. To test this hypothesis, we assessed the capacity of neonatal dendritic cells (DC) to prime and polarize in vitro human naive antigen-specific T cells. We report that mature cord blood DC efficiently prime an oligoclonal population of antigen-specific CD8 T cells, capable of cytolytic activity and IFN-gamma secretion. In contrast, cells primed by immature cord blood DC do not acquire cytolytic activity and secrete lower amounts of IFN-gamma. Upon priming by either immature or mature DC, neonatal T cells acquire markers of activation and differentiation towards effector-memory cells. Our results demonstrate that, if appropriately activated, neonatal DC can prime efficient cytotoxic T lymphocyte (CTL) responses. Furthermore, these findings have important implications for the development of vaccine strategies in early life and for the reconstitution of a functional CTL repertoire after bone marrow transplantation.

Yang JQ, Singh AK, Wilson MT, Satoh M, Stanic AK, Park JJ, Hong S, Gadola SD, Mizutani A, Kakumanu SR et al. 2003. Immunoregulatory role of CD1d in the hydrocarbon oil-induced model of lupus nephritis. J Immunol, 171 (4), pp. 2142-2153. | Show Abstract

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is accompanied by the emergence of autoreactive T cells and a reduction in regulatory T cells. Humans and mice with SLE have reduced numbers of CD1d-restricted NK T cells, suggesting a role for these cells in the regulation of SLE. In this study, we show that CD1d deficiency exacerbates lupus nephritis induced by the hydrocarbon oil pristane. This exacerbation in disease is associated with: 1) reduced TNF-alpha and IL-4 production by T cells, especially during the disease induction phase; and 2) expansion of marginal zone B cells. Strikingly, inoculation of pristane in wild-type mice resulted in reduced numbers and/or functions of NK T cells and CD1d-expressing dendritic cells. These findings suggest that CD1d may play an immunoregulatory role in the development of lupus in the pristane-induced model.

Schultz H, Schinke S, Weiss J, Cerundolo V, Gross WL, Gadola S. 2003. BPI-ANCA in transporter associated with antigen presentation (TAP) deficiency: possible role in susceptibility to Gram-negative bacterial infections. Clin Exp Immunol, 133 (2), pp. 252-259. | Show Abstract | Read more

Although HLA class I expression is diminished in patients with defects in the transporter associated with antigen presentation (TAP), recurrent Gram-negative bacterial lung infections are found from childhood onwards. As MHC class II-mediated responses are normal, other mechanisms that contribute to susceptibility to infections are presumed. The bactericidal/permeability-increasing protein (BPI) is a potent neutrophil antibiotic that neutralizes endotoxin efficiently. As antineutrophil cytoplasmic autoantibodies (ANCA) against BPI were found in the majority of cystic fibrosis patients and correlate with disease severity we examined the prevalence of BPI-ANCA and their contribution to susceptibility to bacterial infections in six TAP-deficient patients. Although only two patients showed ANCA in indirect immunofluorescence, BPI-ANCA occurred in five of six patients in ELISA. Purified IgG from BPI-ANCA-positive sera (five of six) inhibited the antimicrobial function of BPI in vitro. Epitope mapping revealed binding sites not only on the C-terminal but also on the antibiotic N-terminal portion of BPI, indicating that short linear BPI peptide fragments may be long-lived enough to become immunogens. In conclusion, BPI-ANCA are associated strongly with TAP deficiency. Inhibition of the antimicrobial BPI function by BPI-ANCA demonstrates a possible mechanism of how autoantibodies may contribute to increased susceptibility for pulmonary Gram-negative bacterial infections by diminished bacterial clearance.

Marchant A, Appay V, Van Der Sande M, Dulphy N, Liesnard C, Kidd M, Kaye S, Ojuola O, Gillespie GM, Vargas Cuero AL et al. 2003. Mature CD8(+) T lymphocyte response to viral infection during fetal life. J Clin Invest, 111 (11), pp. 1747-1755. | Show Abstract | Read more

Immunization of newborns against viral infections may be hampered by ineffective CD8(+) T cell responses. To characterize the function of CD8(+) T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8(+) T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8(+) T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28(-)CD27(+)CD45RO(+), perforin(low)), and they acquired a late differentiation phenotype (CD28(-)CD27(-)CD45RA(+), perforin(high)) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8(+) T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

Wańkowicz-Kalińska A, van den Wijngaard RM, Tigges BJ, Westerhof W, Ogg GS, Cerundolo V, Storkus WJ, Das PK. 2003. Immunopolarization of CD4+ and CD8+ T cells to Type-1-like is associated with melanocyte loss in human vitiligo. Lab Invest, 83 (5), pp. 683-695. | Show Abstract | Read more

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. High frequencies of melanocyte-reactive cytotoxic T cells in the peripheral blood of vitiligo patients and the observed correlation between perilesional T-cell infiltration and melanocyte loss in situ suggest the important role of cellular autoimmunity in the pathogenesis of this disease. We isolated T cells from both perilesional and nonlesional skin biopsies obtained from five vitiligo patients, then cloned and analyzed their profile of cytokine production after short-term, nonspecific expansion in vitro. Perilesional T-cell clones (TCC) derived from patients with vitiligo exhibited a predominant Type-1-like cytokine secretion profile, whereas the degree of Type-1 polarization in uninvolved skin-derived TCC correlated with the process of microscopically observed melanocyte destruction in situ. Detailed analysis of broad spectrum of cytokines produced by perilesional- and nonlesional-derived CD4+ and CD8+ TCC confirmed polarization toward Type-1-like in both CD4 and CD8 compartments, which paralleled depigmentation process observed locally in the skin. Furthermore, CD8+ TCC derived from two patients also were analyzed for reactivity against autologous melanocytes. The antimelanocyte cytotoxic reactivity was observed among CD8+ TCC isolated from perilesional biopsies of two patients with vitiligo. Finally, in two of five patients, tetramer analysis revealed presence of high frequencies of Mart-1-specific CD8 T cells in T-cell lines derived from perilesional skin. Altogether our data support the role of cellular mechanisms playing a significant part in the destruction of melanocytes in human autoimmune vitiligo.

Jones E, Price DA, Dahm-Vicker M, Cerundolo V, Klenerman P, Gallimore A. 2003. The influence of macrophage inflammatory protein-1alpha on protective immunity mediated by antiviral cytotoxic T cells. Immunology, 109 (1), pp. 68-75. | Show Abstract | Read more

Macrophage inflammatory protein 1alpha (MIP-1alpha), a member of the CC-chemokine subfamily, is known to induce chemotaxis of a variety of cell types in vivo. Although the role of MIP-1alpha in inflammatory responses generated following primary infection of mice with many different pathogens has been characterized, the influence of this chemokine on the generation of antigen-specific T-cell responses in vivo is less well understood. This is important, as virus-specific CD8+ T lymphocytes (CTL) play a crucial role in defence against viral infections, both acutely and in the long term. In this study, we compared the ability of wild-type and MIP-1alpha-deficient (MIP-1alpha-/-) mice to mount CTL responses specific for the immunodominant epitope derived from influenza nucleoprotein (NP366-374). Influenza-specific CTL responses were compared with respect to frequency, cytotoxic activity and ability to clear subsequent infections with recombinant vaccinia viruses expressing the influenza NP. The results indicate that antiviral CTL generated in MIP-1alpha-/- mice are slightly impaired in their ability to protect against a subsequent infection. However, impaired in vivo CTL-mediated antiviral protection was found to be associated with reduced cytotoxicity rather than with a failure of the CTL to migrate to peripheral sites of infection.

Salio M, Cella M, Vermi W, Facchetti F, Palmowski MJ, Smith CL, Shepherd D, Colonna M, Cerundolo V. 2003. Plasmacytoid dendritic cells prime IFN-gamma-secreting melanoma-specific CD8 lymphocytes and are found in primary melanoma lesions. Eur J Immunol, 33 (4), pp. 1052-1062. | Show Abstract | Read more

Plasmacytoid dendritic cells (PDC) are a small population of leukocytes specialized in the production of type I IFN. It has been shown that PDC have a potent T cell stimulatory capacity in allogeneic mixed lymphocyte reaction, However, their role in initiating primary immune responses remains elusive. We report that blood PDC efficiently prime naive CD8(+) lymphocytes specific for the melan-A(26-35) epitope to become IFN-gamma producing cells in vitro. In addition, we found that CD40L-stimulated PDC induce expression on primed melan-A-specific T cells of cutaneous lymphocyte antigen and L-selectin (CD62L), homing receptors that allow the migration of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri-tumoral area of most primary cutaneous melanomas in vivo and that type I IFN-containing supernatants derived from PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro. Our results suggest a new immunomodulatory role for tissue infiltrating PDC, which may prime tumor-specific T cell responses and affect tumor growth via soluble factors.

Lucas M, Gadola S, Meier U, Young NT, Harcourt G, Karadimitris A, Coumi N, Brown D, Dusheiko G, Cerundolo V, Klenerman P. 2003. Frequency and phenotype of circulating Valpha24/Vbeta11 double-positive natural killer T cells during hepatitis C virus infection. J Virol, 77 (3), pp. 2251-2257. | Show Abstract | Read more

Natural killer T (NKT) cells are thought to be involved in innate responses against infection. We investigated one specific type of NKT cell, Valpha24/Vbeta11 double positive, in hepatitis C virus (HCV) infection. Lower frequencies of this population were detected in the blood of HCV PCR-positive patients than in controls. Unlike Valpha24/Vbeta11 NKT cells found in blood, those in the liver appeared to be recently activated.

Lautscham G, Haigh T, Mayrhofer S, Taylor G, Croom-Carter D, Leese A, Gadola S, Cerundolo V, Rickinson A, Blake N. 2003. Identification of a TAP-independent, immunoproteasome-dependent CD8+ T-cell epitope in Epstein-Barr virus latent membrane protein 2. J Virol, 77 (4), pp. 2757-2761. | Show Abstract | Read more

We have identified an HLA-A2-restricted CD8(+) T-cell epitope, FLYALALLL, in the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2), an important target antigen in the context of EBV-associated malignancies. This epitope is TAP independent, like other hydrophobic LMP2-derived epitopes, but uniquely is dependent upon the immunoproteasome for its generation.

Simon AK, Gallimore A, Jones E, Sawitzki B, Cerundolo V, Screaton GR. 2002. Fas ligand breaks tolerance to self-antigens and induces tumor immunity mediated by antibodies. Cancer Cell, 2 (4), pp. 315-322. | Show Abstract | Read more

The role of Fas ligand (FasL) in programmed cell death via interaction with its receptor Fas is well characterized. It has been proposed that expression of FasL can confer immune privilege to some organs, allowing them to kill infiltrating lymphocytes and inflammatory cells. However, a number of studies have shown that when tumors or transplants express FasL, rejection often occurs as a consequence of proinflammatory functions of FasL. Here we demonstrate that FasL elicits tumor immunity in a murine melanoma model with weak immunogenicity and low expression of major histocompatibility complex (MHC) class I. We show that protected mice recognize melanocyte differentiation self-antigens. Importantly, tumor immunity is mediated by antibodies, as it can be transferred by serum from protected mice.

Choi EM, Palmowski M, Chen J, Cerundolo V. 2002. The use of chimeric A2K(b) tetramers to monitor HLA A2 immune responses in HLA A2 transgenic mice. J Immunol Methods, 268 (1), pp. 35-41. | Show Abstract | Read more

HLA A2 (A2) transgenic mice are currently being used to compare different vaccination protocols. However, the monitoring of A2 restricted CTL in A2 transgenic mice have been hampered by poor staining efficiency of mouse CTL by A2 tetramers. We demonstrate here that chimeric A2 tetramers containing mouse H-2K(b) (K(b)) alpha3 domain (A2K(b) tetramers) can be used as staining reagents to monitor A2 restricted CTL responses in A2 transgenic mice. The increased ability of A2K(b) tetramers to stain mouse A2 restricted CTL, as compared with A2 tetramers, correlated with their higher binding affinity for mouse A2 restricted CTL. The use of these novel staining reagents will allow efficient comparison of vaccination strategies and rapid identification of novel CTL epitopes in A2 transgenic mice.

Palmowski M, Salio M, Dunbar RP, Cerundolo V. 2002. The use of HLA class I tetramers to design a vaccination strategy for melanoma patients. Immunol Rev, 188 (1), pp. 155-163. | Show Abstract | Read more

Progress in human tumor immunology has recently been accelerated by new assays for antigen-specific cytotoxic T lymphocytes (CTLs). We have used tetrameric MHC class I complexes (tetramers) to study melanoma-specific CTLs both in vivo and in vitro, and have utilized the results to optimize vaccination strategies for patients. Tetramers have provided some of the best evidence to date that CTL responses against melanoma antigens arise spontaneously in patients. However, CTL responses to common (nonmutated) melanoma epitopes are generally weak or localized, and occur mostly in advanced metastatic disease, hence justifying early immunotherapeutic approaches. These observations led us to design a polyvalent vaccine construct for early administration to melanoma patients at high risk of progression. To compare possible vaccination protocols, we encoded this construct in several different vectors, and developed novel tetramers to track responses to the human melanoma epitopes in transgenic mice. Priming and boosting with the same poly-epitope construct encoded in heterologous vectors led to the expansion of CTLs with a single dominant specificity. Separating the antigens for independent presentation by antigen-presenting cells reversed the effect of immunodominance and induced a powerful polyvalent CTL response. These results provide important pointers for future vaccination trials, and tetramers will form an important tool in the immunomonitoring of these clinical studies.

Gadola SD, Zaccai NR, Harlos K, Shepherd D, Castro-Palomino JC, Ritter G, Schmidt RR, Jones EY, Cerundolo V. 2002. Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains. Nat Immunol, 3 (8), pp. 721-726. | Show Abstract | Read more

The human genome encodes five nonpolymorphic major histocompatibility complex class I-like glycoproteins, CD1a to CD1e, that present lipid antigens for specific recognition by T lymphocytes. Using single alkyl chain detergents, we developed a protocol to generate recombinant human CD1b-lipid complexes. We present here the crystal structures of CD1b in complex with either phosphatidylinositol or ganglioside GM2 at 2.3 A and 2.8 A resolutions, respectively. The antigen-binding groove houses four interlinked hydrophobic channels that are occupied by the alkyl chains of the glycolipid plus two detergent molecules. A distinct exit beneath the alpha 2 helix further contributes to the plasticity of the binding groove. These structures reveal the mechanism by which two alkyl chain lipids bind to CD1b, and how CD1b can adapt to ligands of different alkyl chain length. They also suggest how very long alkyl chains, such as those of mycolic acid, could be fully contained within the binding groove. These results extend the spectrum of potential CD1b ligands by revealing that, in addition to two alkyl chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding groove.

Smith DC, Gallimore A, Jones E, Roberts B, Lord JM, Deeks E, Cerundolo V, Roberts LM. 2002. Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation. J Immunol, 169 (1), pp. 99-107. | Show Abstract

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.

Glick M, Price DA, Vuidepot AL, Andersen TB, Hutchinson SL, Laugel B, Sewell AK, Boulter JM, Dunbar PR, Cerundolo V et al. 2002. Novel CD8+ T cell antagonists based on beta 2-microglobulin. J Biol Chem, 277 (23), pp. 20840-20846. | Show Abstract | Read more

The CD8 coreceptor of cytotoxic T lymphocytes binds to a conserved region of major histocompatibility complex class I molecules during recognition of peptide-major histocompatibility complex (MHC) class I antigens on the surface of target cells. This event is central to the activation of cytotoxic T lymphocyte (CTL) effector functions. The contribution of the MHC complex class I light chain, beta(2)-microglobulin, to CD8alphaalpha binding is relatively small and is mediated mainly through the lysine residue at position 58. Despite this, using molecular modeling, we predict that its mutation should have a dramatic effect on CD8alphaalpha binding. The predictions are confirmed using surface plasmon resonance binding studies and human CTL activation assays. Surprisingly, the charge-reversing mutation, Lys(58) --> Glu, enhances beta(2)m-MHC class I heavy chain interactions. This mutation also significantly reduces CD8alphaalpha binding and is a potent antagonist of CTL activation. These results suggest a novel approach to CTL-specific therapeutic immunosuppression.

Gadola SD, Dulphy N, Salio M, Cerundolo V. 2002. Valpha24-JalphaQ-independent, CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes. J Immunol, 168 (11), pp. 5514-5520. | Show Abstract

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.

Palmowski MJ, Choi EM, Hermans IF, Gilbert SC, Chen JL, Gileadi U, Salio M, Van Pel A, Man S, Bonin E et al. 2002. Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol, 168 (9), pp. 4391-4398. | Show Abstract

Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2K(b) H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.

Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, Ogg GS, King A, Lechner F, Spina CA et al. 2002. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med, 8 (4), pp. 379-385. | Show Abstract | Read more

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

Klenerman P, Cerundolo V, Dunbar PR. 2002. Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol, 2 (4), pp. 263-272. | Show Abstract | Read more

To understand the success or failure of immune responses against pathogens or tumours requires the direct measurement of specific lymphocytes. Recently, there has been an explosion of data in this field through the use of several new tools for measuring the number and function of T cells. This has allowed immunologists who study human disease and mouse models of infection and cancer to readily track specific T cells--in both time and space. Although there are common patterns, over time, each host-pathogen relationship seems to develop unique characteristics, as reflected in the quality of the T-cell response.

Jones E, Dahm-Vicker M, Simon AK, Green A, Powrie F, Cerundolo V, Gallimore A. 2002. Depletion of CD25+ regulatory cells results in suppression of melanoma growth and induction of autoreactivity in mice. Cancer Immun, 2 pp. 1. | Show Abstract

Treatment with monoclonal antibodies (mAbs) specific for CD25 (anti-CD25 mAb) has been shown to suppress growth of a variety of different tumours in mice. These studies did not however determine whether or not anti-CD25 mAbs facilitate tumour rejection by depletion of regulatory T cells or by binding to tumour-specific effector cells. Using a murine model of melanoma we have found that treatment of mice with anti-CD25 mAb facilitates long-term CD4+ T cell-mediated tumour immunity through depletion of CD25+ regulatory cells. We further show that the effector CD4+ T cells confer long-term tumour immunity even in the presence of CD25+ regulatory cells and do not require CD8+ T cells for tumour rejection. The inhibitory impact of anti-CD25 mAb treatment on tumour growth may be the result of depleting CD25+ regulatory cells that normally inhibit the generation of immune responses to self-antigens that are shared by the tumour. We have performed experiments to determine whether or not immune responses to melanocyte antigens are generated in anti-CD25 mAb-treated, melanoma-immune mice. The results of the experiments indicate that a T cell response to the melanocyte antigen tyrosinase accompanies suppression of tumour growth in mice lacking CD25+ regulatory cells.

Sewell AK, Booth BL, Cerundolo V, Phillips RE, Price DA. 2002. Differential processing of HLA A2-restricted HIV type 1 cytotoxic T lymphocyte epitopes. Viral Immunol, 15 (1), pp. 193-196. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) play a key role in the control of persistent viral infections. Differences in the quality of this cellular immune response influence the long-term outcome of such infections, but the factors that determine which virus-derived peptide epitopes are targeted by CTLs remain poorly understood. Here, we examine the antigen-processing requirements of three human leukocyte antigen (HLA) A*0201-restricted HIV-1 CTL epitopes. Each of these three peptides appears to be generated by a distinct proteolytic pathway, despite presentation on the cell surface in association with the same HLA class I molecule. Presentation of the commonly immunodominant SLYNTVATL (HIV-1 p17 Gag; residues 77-85) epitope was unaffected by inhibition of the proteasome with lactacystin, but was dependent on the presence of the beta-subunit LMP7. These findings are consistent with emerging data on the complexity of peptide epitope generation, and suggest that differences in antigen processing might contribute to patterns of CTL recognition in vivo.

Smith CL, Dulphy N, Salio M, Cerundolo V. 2002. Immunotherapy of colorectal cancer. Br Med Bull, 64 (1), pp. 181-200. | Show Abstract | Read more

Over the last decade, there has been a rapid expansion in the field of tumour immunology. There is now convincing evidence that both the cellular and humoral arms of the immune system are capable of interacting with tumour cells. The most significant advances have been in our understanding of cellular responses and the complex events that lead to T-lymphocyte activation, as well as in the identification of tumour antigens recognised by T-lymphocytes. This knowledge has led to the development of anticancer immunotherapies designed to produce tumour antigen-specific T-cell responses, adding to the earlier antibody or whole-cell vaccine approaches. In addition, new methods have been developed to quantify antigen-specific T-cell responses, and the emergent field of recombinant gene technology has led to an increasing number of novel methods for vaccine delivery. This review will explore these advances, as well as possible future directions, with an emphasis on colorectal cancer.

Evans M, Borysiewicz LK, Evans AS, Rowe M, Jones M, Gileadi U, Cerundolo V, Man S. 2001. Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6. J Immunol, 167 (9), pp. 5420-5428. | Show Abstract

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.

Hemelaar J, Bex F, Booth B, Cerundolo V, McMichael A, Daenke S. 2001. Human T-cell leukemia virus type 1 Tax protein binds to assembled nuclear proteasomes and enhances their proteolytic activity. J Virol, 75 (22), pp. 11106-11115. | Show Abstract | Read more

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates the HTLV-1 long terminal repeat and key regulatory proteins involved in inflammation, activation, and proliferation and may induce cell transformation. Tax is also the immunodominant target antigen for cytotoxic T cells in HTLV-1 infection. We found that Tax bound to assembled nuclear proteasomes, but Tax could not be detected in the cytoplasm. Confocal microscopy revealed a partial colocalization of Tax with nuclear proteasomes. As Tax translocated into the nucleus very quickly after synthesis, this process probably takes place prior to and independent of proteasome association. Tax mutants revealed that both the Tax N and C termini play a role in proteasome binding. We also found that proteasomes from Tax-transfected cells had enhanced proteolytic activity on prototypic peptide substrates. This effect was not due to the induction of the LMP2 and LMP7 proteasome subunits. Furthermore, Tax appeared to be a long-lived protein, with a half-life of around 15 h. These data suggest that the association of Tax with the proteasome and the enhanced proteolytic activity do not target Tax for rapid degradation and may not determine its immunodominance.

Smith C, Cerundolo V. 2001. Immunotherapy of melanoma. Immunology, 104 (1), pp. 1-7. | Read more

Salio M, Shepherd D, Dunbar PR, Palmowski M, Murphy K, Wu L, Cerundolo V. 2001. Mature dendritic cells prime functionally superior melan-A-specific CD8+ lymphocytes as compared with nonprofessional APC. J Immunol, 167 (3), pp. 1188-1197. | Show Abstract

Priming of melan-A(26/27-35)-specific CTL occurs only in a fraction of late stage melanoma patients, whereas during the early stages of the disease and in healthy volunteers, melan-A CTL have functional and phenotypic markers consistent with a naive phenotype. To study the requirements for expansion of naive melan-A CTL from healthy donors, we set up an in vitro priming protocol and, using tetramer assays, we demonstrate that the activity and phenotype of the expanded melan-A CTL are profoundly influenced by the type of APC used. Priming by nonprofessional APC leads to expansion of melan-A CTL with reduced cytolytic activity and low level of IFN-gamma secretion. In contrast, mature dendritic cells (DC) expand cytolytic and IFN-gamma-producing melan-A CTL. Priming by mature DC is also efficient at low peptide concentration and requires only one round of stimulation. Finally, we observed that a significant fraction of CD45RO(+) melan-A CTL primed by mature DC expresses high levels of the homing receptor CD62L, whereas CTL primed by nonprofessional APC express CD62L in lower percentages and at lower levels. These results suggest that suboptimal priming by nonprofessional APC could account for the presence in vivo of dysfunctional cells and strongly support the immunotherapeutic use of mature DC for expansion of effector and memory Ag-specific CTL.

Kelleher AD, Booth BL, Sewell AK, Oxenius A, Cerundolo V, McMichael AJ, Phillips RE, Price DA. 2001. Effects of retroviral protease inhibitors on proteasome function and processing of HIV-derived MHC class I-restricted cytotoxic T lymphocyte epitopes. AIDS Res Hum Retroviruses, 17 (11), pp. 1063-1066. | Read more

Propato A, Cutrona G, Francavilla V, Ulivi M, Schiaffella E, Landt O, Dunbar R, Cerundolo V, Ferrarini M, Barnaba V. 2001. Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming. Nat Med, 7 (7), pp. 807-813. | Show Abstract | Read more

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

Xu XN, Purbhoo MA, Chen N, Mongkolsapaya J, Cox JH, Meier UC, Tafuro S, Dunbar PR, Sewell AK, Hourigan CS et al. 2001. A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex. Immunity, 14 (5), pp. 591-602. | Show Abstract | Read more

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

Karadimitris A, Gadola S, Altamirano M, Brown D, Woolfson A, Klenerman P, Chen JL, Koezuka Y, Roberts IA, Price DA et al. 2001. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc Natl Acad Sci U S A, 98 (6), pp. 3294-3298. | Show Abstract | Read more

CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipid specific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone- and foldase-assisted refolding of denatured CD1d molecules and beta(2)-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-alpha-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

Dunbar PR, Smith CL, Chao D, Salio M, Shepherd D, Mirza F, Lipp M, Lanzavecchia A, Sallusto F, Evans A et al. 2000. A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response. J Immunol, 165 (11), pp. 6644-6652. | Show Abstract

In a significant proportion of melanoma patients, CTL specific for the melan-A(26/7-35) epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer(+) cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer(+) cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7(+) CD45R0(-) CD45RA(+) phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer(+) cells were shifted toward a CCR7(-) CD45R0(+) CD45RA(-) phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

Oelke M, Kurokawa T, Hentrich I, Behringer D, Cerundolo V, Lindemann A, Mackensen A. 2000. Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology. Scand J Immunol, 52 (6), pp. 544-549. | Show Abstract | Read more

Cell therapy with antigen-specific T cells holds promise for various diseases including cancer and viral infections. The powerful enrichment procedure based on major histocompatibility complex (MHC)-tetramers, however, is of limited applicability so far. Therefore, the recently developed cell surface affinity matrix technology that allows direct identification and enrichment of life antigen-specific T cells based on cytokine secretion was evaluated in this respect. To this end, CD8(+) T cells directed against the HLA-A(*)0201-restricted melanoma-associated peptide Melan-A (aa26-35) were generated by combining stimulation of peptide-pulsed autologous dendritic cells (DC) with antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies (MoAb). Antigen-specific cytotoxic T lymphocyte (CTL) were detected based on stimulation-induced interferon (IFN)-gamma and interleukin (IL)-4 secretion and enriched > 100-fold using the cell surface affinity matrix technology. The resulting IFN-gamma- and IL-4-secreting CTL lines contained > 80% and > 70% cytokine positive T cells, respectively. They exhibited a cytotoxic activity against Melan-A expressing target cells that was significantly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A-tetramers revealed a high correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that cytokine-driven enrichment based on the cell surface affinity matrix technology enables selective isolation of functionally active antigen-specific CTL that may be used for an adoptive T cell transfer in immunotherapy.

Oelke M, Vogl S, Cerundolo V, Andreesen R, Mackensen A. 2000. Generation and enrichment of antigen-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy. BLOOD, 96 (11), pp. 28A-28A.

Bellantuono I, Macchiarulo E, Gao LQ, Dazzi F, Cerundolo V, Marley SB, Gordon MY, Goldman JM, Stauss HJ. 2000. Immunoresponse to Wilms Tumor antigen-1 (WT-1) in CML patients. BLOOD, 96 (11), pp. 144A-144A.

Gnjatic S, Nagata Y, Jager E, Stockert E, Shankara S, Roberts BL, Mazzara GP, Lee SY, Dunbar PR, Dupont B et al. 2000. Strategy for monitoring T cell responses to NY-ESO-1 in patients with any HLA class I allele. Proc Natl Acad Sci U S A, 97 (20), pp. 10917-10922. | Show Abstract | Read more

NY-ESO-1 elicits frequent antibody responses in cancer patients, accompanied by strong CD8(+) T cell responses against HLA-A2-restricted epitopes. To broaden the range of cancer patients who can be assessed for immunity to NY-ESO-1, a general method was devised to detect T cell reactivity independent of prior characterization of epitopes. A recombinant adenoviral vector encoding the full cDNA sequence of NY-ESO-1 was used to transduce CD8-depleted peripheral blood lymphocytes as antigen-presenting cells. These modified antigen-presenting cells were then used to restimulate memory effector cells against NY-ESO-1 from the peripheral blood of cancer patients. Specific CD8(+) T cells thus sensitized were assayed on autologous B cell targets infected with a recombinant vaccinia virus encoding NY-ESO-1. Strong polyclonal responses were observed against NY-ESO-1 in antibody-positive patients, regardless of their HLA profile. Because the vectors do not cross-react immunologically, only responses to NY-ESO-1 were detected. The approach described here allows monitoring of CD8(+) T cell responses to NY-ESO-1 in the context of various HLA alleles and has led to the definition of NY-ESO-1 peptides presented by HLA-Cw3 and HLA-Cw6 molecules.

Schuler-Thurner B, Dieckmann D, Keikavoussi P, Bender A, Maczek C, Jonuleit H, Röder C, Haendle I, Leisgang W, Dunbar R et al. 2000. Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells. J Immunol, 165 (6), pp. 3492-3496. | Show Abstract

Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 x 106 s.c. followed by two i.v. ones of 6 and 12 x 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2. 1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-gamma-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.

Appay V, Dunbar PR, Cerundolo V, McMichael A, Czaplewski L, Rowland-Jones S. 2000. RANTES activates antigen-specific cytotoxic T lymphocytes in a mitogen-like manner through cell surface aggregation. Int Immunol, 12 (8), pp. 1173-1182. | Show Abstract | Read more

RANTES (regulated upon activation, normal T cell expressed and secreted) is released by cytotoxic T lymphocytes (CTL), and is a potent chemoattractant factor for monocytes and T cells, also known for its ability to suppress HIV infection. At micromolar concentration, RANTES is able to activate leukocytes, and, paradoxically, to enhance HIV infection in vitro. These latter properties are dependent on its ability to self-aggregate. In order to understand further the mechanism of RANTES-induced activation, the effects of both aggregated and disaggregated RANTES on antigen-specific CD8(+) clones were studied in comparison with the effects of specific antigens and in the presence of specific inhibitors of RANTES-mediated activation. We observed large amounts of RANTES aggregated on the cell surface, which led to cell activation, including up-regulation of cell surface markers, and secretion of IFN-gamma and macrophage inflammatory protein (MIP)-1beta. Specific inhibitors of RANTES-induced activation, such as soluble glycosaminoglycans, MIP-1alpha and MIP-1beta, acted by preventing the binding of RANTES on the cell surface. These studies suggest that RANTES acted more like a mitogen than an antigen-independent activator.

Larsson M, Messmer D, Somersan S, Fonteneau JF, Donahoe SM, Lee M, Dunbar PR, Cerundolo V, Julkunen I, Nixon DF, Bhardwaj N. 2000. Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells. J Immunol, 165 (3), pp. 1182-1190. | Show Abstract

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.

Gadola SD, Moins-Teisserenc HT, Trowsdale J, Gross WL, Cerundolo V. 2000. TAP deficiency syndrome. Clin Exp Immunol, 121 (2), pp. 173-178. | Read more

Chen JL, Dunbar PR, Gileadi U, Jäger E, Gnjatic S, Nagata Y, Stockert E, Panicali DL, Chen YT, Knuth A et al. 2000. Identification of NY-ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL. J Immunol, 165 (2), pp. 948-955. | Show Abstract

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157-165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157-165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157-165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.

Mortarini R, Borri A, Tragni G, Bersani I, Vegetti C, Bajetta E, Pilotti S, Cerundolo V, Anichini A. 2000. Peripheral burst of tumor-specific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8+ T cells in melanoma patients receiving interleukin 12. Cancer Res, 60 (13), pp. 3559-3568. | Show Abstract

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-35)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

Valmori D, Dutoit V, Liénard D, Lejeune F, Speiser D, Rimoldi D, Cerundolo V, Dietrich PY, Cerottini JC, Romero P. 2000. Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients. J Immunol, 165 (1), pp. 533-538. | Show Abstract

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.

Oelke M, Moehrle U, Chen JL, Behringer D, Cerundolo V, Lindemann A, Mackensen A. 2000. Generation and purification of CD8+ melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy. Clin Cancer Res, 6 (5), pp. 1997-2005. | Show Abstract

Tumor antigens that might serve as potential targets for adoptive T-cell therapy have been defined in different tumor entities, especially in malignant melanoma. To generate conditions to induce primary T-cell responses against different HLA-A*0201-restricted melanoma peptides and to allow further expansion of peptide-specific T cells for adoptive transfer, CD8+-purified T cells from healthy donors were stimulated with Melan-A-pulsed autologous dendritic cells. Dendritic cells were generated in vitro from monocytes with granulocyte macrophage colony-stimulating factor, interleukin-4, and transforming growth factor-beta1. After 3-4 weekly stimulation cycles with Melan-A-pulsed DCs, we were able to induce a strong peptide-specific CTL response in vitro. MHC-peptide tetramer staining revealed a frequency of up to 3.5% CD8+/Melan-A+ T cells. Additional antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies together with interleukin-2 gave rise to 600-fold expansion of CD8+ CTLs that maintained Melan-A specificity and were able to efficiently lyse Melan-A-expressing melanoma cells. To enrich antigen-specific T cells in vitro, we used a recently established technology for analysis and sorting of live cells according to secreted cytokines. In the present study, we demonstrated that Melan-A-specific T cells can be purified by magnetic separation according to secreted IFN-gamma. These cells revealed a very potent monospecific CTL response, even at low E:T ratios, against Melan-A-pulsed and Melan-A-expressing target cells. Altogether, our study demonstrated that we have developed an efficient method for generating large numbers of peptide-specific T cells in vitro that may be used for adoptive T-cell transfer in tumor immunotherapy.

Mackensen A, Herbst B, Chen JL, Köhler G, Noppen C, Herr W, Spagnoli GC, Cerundolo V, Lindemann A. 2000. Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generated in vitro from CD34(+) hematopoietic progenitor cells. Int J Cancer, 86 (3), pp. 385-392. | Show Abstract | Read more

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34(+) peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-alpha. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1(+) or HLA-A2(+) patients received at least 4 i.v. infusions of 5 x 10(6) to 5 x 10(7) DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I-II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2(+) patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.

Jäger E, Nagata Y, Gnjatic S, Wada H, Stockert E, Karbach J, Dunbar PR, Lee SY, Jungbluth A, Jäger D et al. 2000. Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral and cellular immune responses. Proc Natl Acad Sci U S A, 97 (9), pp. 4760-4765. | Show Abstract | Read more

NY-ESO-1, a member of the cancer-testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic CD8(+) T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure CD8(+) T cell responses to NY-ESO-1, (ii) to determine the frequency of CD8(+) T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between CD8(+) T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. CD8(+) T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO-1 antibody, but not in patients lacking antibody or in patients with NY-ESO-1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines.

Ogg GS, Dunbar PR, Cerundolo V, McMichael AJ, Lemoine NR, Savage P. 2000. Sensitization of tumour cells to lysis by virus-specific CTL using antibody-targeted MHC class I/peptide complexes. Br J Cancer, 82 (5), pp. 1058-1062. | Show Abstract | Read more

A number of cell surface molecules with specificity to tumour cells have been identified and monoclonal antibodies (mAb) to some of these antigens have been used for targeting tumour cells in vivo. We have sought to link the powerful effector mechanisms of cytotoxic T-cells with the specificity of mAb, by targeting recombinant HLA class I molecules to tumour cells using an antibody delivery system. Soluble recombinant MHC class I/peptide complexes including HLA-A2.1 refolded around an immunodominant peptide from the HIV gag protein (HLA-A2/gag) were synthesized, and the stability of these complexes at 37 degrees C was confirmed by enzyme-linked immunosorbent assay using a conformation-specific antibody. MHC class I-negative lymphoma cells (Daudi) were labelled with a biotinylated mAb specific for a cell surface protein (anti-CD20) then linked to soluble biotinylated HLA-A2/gag complexes using an avidin bridge. Flow cytometry revealed strong labelling of lymphoma cells with HLA-A2/gag complexes (80-fold increase in mean channel fluorescence). CTL specific for HLA-A2/gag efficiently lysed complex-targeted cells, while control CTL (specific for an HLA-A2.1-restricted epitope of melan-A) did not. Similarly, SK-mel-29 melanoma cells were also efficiently lysed by HLA-A2/gag-specific CTL when HLA-A2/gag complexes were linked to their surface via the HMW-MAA specific anti-melanoma antibody 225.28s. With further consideration to the in vivo stability of the MHC class I/peptide complexes, this system could prove a new strategy for the immunological therapy of cancer.

Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, Gadola S, Cerundolo V, Borysiewicz LK, McMichael AJ, Wilkinson GW. 2000. Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science, 287 (5455), pp. 1031. | Show Abstract | Read more

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

Salio M, Cerundolo V, Lanzavecchia A. 2000. Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells. Eur J Immunol, 30 (2), pp. 705-708. | Show Abstract | Read more

To identify environmental stimuli that induce dendritic cell (DC) maturation, we exposed human monocyte-derived immature DC to apoptotic or necrotic cells and measured the levels of expression of costimulatory molecules and cytokine production. While most necrotic or apoptotic cells did not have any effect, some induced DC maturation as detected by up-regulation of CD83 and B7.2 and production of IL-12 and IL-6. The capacity of these cell lines to induce DC maturation was due to their contamination by mycoplasma, since the maturation-inducing effect disappeared when the cells were treated with cyproxin. Furthermore, cell lines deliberately infected with mycoplasma containing supernatant acquired the capacity to induce DC maturation. Our results reveal that DC are able to sense mycoplasma infection and mature as they do in response to most viruses and bacteria. In contrast, apoptotic or necrotic cells fail to induce DC maturation.

Valmori D, Lévy F, Miconnet I, Zajac P, Spagnoli GC, Rimoldi D, Liénard D, Cerundolo V, Cerottini JC, Romero P. 2000. Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue. J Immunol, 164 (2), pp. 1125-1131. | Show Abstract

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Youde SJ, Dunbar PR, Evans EM, Fiander AN, Borysiewicz LK, Cerundolo V, Man S. 2000. Use of fluorogenic histocompatibility leukocyte antigen-A*0201/HPV 16 E7 peptide complexes to isolate rare human cytotoxic T-lymphocyte-recognizing endogenous human papillomavirus antigens. Cancer Res, 60 (2), pp. 365-371. | Show Abstract

Cervical cancer (CaCx) is the second most common female malignancy worldwide and remains a clinical problem despite improvements in early detection and therapy. CaCx and preinvasive cervical intraepithelial neoplasia (CIN3) are strongly associated with infection by human papillomavirus (HPV), particularly types 16 and 18. Two nonstructural viral proteins, E6 and E7, are constitutively expressed in cervical tumors and are crucial for the maintenance of the transformed phenotype. These proteins thus provide attractive targets for immunotherapy of CaCx mediated by CD8+ CTLs. However, reliable detection and generation of HPV-specific CTLs in humans has been difficult. Recently, soluble fluorogenic MHC-peptide complexes (tetramers) have greatly increased the sensitivity of antiviral and antitumor CTL detection. To examine the feasibility of this approach for detecting HPV-specific CTLs, we constructed a tetramer consisting of HLA-A*0201 and the best studied HPV CTL peptide epitope, HPV 16 E711-20. Between 2 and 12% of short-term HPV 16 E711-2 CTL lines derived from CaCx patients stained highly with the tetramer. Direct ex vivo staining of peripheral blood mononuclear cells revealed CD8+ tetramer+ high cells at low frequencies in both CIN3 patients (1 of 1,260 to 1 of 19,073) and normal controls (1 of 1,855 to 1 of 42,004). However, short-term in vitro stimulation with the HPV 16 E711-20 peptide expanded CD8+tetramer+ cells to a greater extent in the peripheral blood mononuclear cells from CIN3 patients. Furthermore, the tetramer provided a powerful tool to isolate polyclonal and clonal peptide-specific CTLs from an established HPV 16 E7,11-20-specific CTL line. These purified CTLs were able to lyse both peptide-pulsed targets and targets expressing endogenously processed HPV antigens. This tetramer may therefore be useful for selecting rare high-affinity HPV-specific CTLs for the immunotherapy of CaCx.

Romero P, Pittet MJ, Valmori D, Speiser DE, Cerundolo V, Liénard D, Lejeune F, Cerottini JC. 2000. Immune monitoring in cancer immunotherapy. Ernst Schering Res Found Workshop, 30 (30), pp. 75-97.

Cerundolo V. 2000. Use of major histocompatibility complex class I tetramers to monitor tumor-specific cytotoxic T lymphocyte response in melanoma patients. Cancer Chemother Pharmacol, 46 Suppl (S1), pp. S83-S85. | Show Abstract | Read more

There is now considerable evidence that human tumors often express antigens that render them susceptible to lysis by cytotoxic T lymphocytes (CTLs). These findings have raised hope for the development of cancer vaccines to trigger a tumor-specific immune response in cancer patients. To optimize the immunogenicity of cancer vaccines, it is important to improve the monitoring of the immune response. The use of tetrameric soluble major histocompatibility complex (MHC) class I/peptide complexes ("tetramers") to identify tumor-specific CTLs has shown that these novel reagents allow rapid and accurate analysis of human CTL responses in cancer patients. We have used fluorescence-driven cell sorting to clone tumor-specific CTLs after staining with tetrameric MHC class I/peptide complexes. Analysis of melanoma-infiltrated lymph nodes revealed that strong CTL responses often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Gileadi U, Moins-Teisserenc HT, Correa I, Booth BL, Dunbar PR, Sewell AK, Trowsdale J, Phillips RE, Cerundolo V. 1999. Generation of an immunodominant CTL epitope is affected by proteasome subunit composition and stability of the antigenic protein. J Immunol, 163 (11), pp. 6045-6052. | Show Abstract

Generation of the HLA-A0201 (A2) influenza Matrix 58-66 epitope contained within the full-length Matrix protein is impaired in cells lacking the proteasome subunits low molecular protein 2 (LMP2) and LMP7. This Ag presentation block can be relieved by transfecting the wild-type LMP7 cDNA into LMP7-deficient cells. A mutated form of LMP7, lacking the two threonines at the catalytic active site, was equally capable of relieving the block in presentation of the influenza Matrix A2 epitope. These observations were extended by analyzing whether modification of the influenza Matrix protein could overcome the block in presentation of the A2 Matrix epitope. Expression of either a rapidly degraded form of the full-length Matrix protein or shorter Matrix fragments led to an efficient presentation of the A2 influenza Matrix epitope by LMP7-negative cells. These findings demonstrate two main points: 1) LMP7 incorporation into the proteasome is of greater importance for the generation of the influenza A2 Matrix epitope than the presence of the LMP7's catalytic site; and 2) the interplay between cytosolic proteases and stability of target proteins is of importance in optimization of Ag presentation. These observations may have relevance to the immunodominance of tumor and viral epitopes and raise the possibility that generation of shorter protein fragments could be a mechanism to ensure optimal Ag presentation by cells expressing low levels of LMP7.

Valmori D, Fonteneau JF, Valitutti S, Gervois N, Dunbar R, Liénard D, Rimoldi D, Cerundolo V, Jotereau F, Cerottini JC et al. 1999. Optimal activation of tumor-reactive T cells by selected antigenic peptide analogues. Int Immunol, 11 (12), pp. 1971-1980. | Show Abstract

Many mechanisms have been proposed to explain why immune responses against human tumor antigens are generally ineffective. For example, tumor cells have been shown to develop active immune evasion mechanisms. Another possibility is that tumor antigens are unable to optimally stimulate tumor-specific T cells. In this study we have used HLA-A2/Melan-A peptide tetramers to directly isolate antigen-specific CD8(+) T cells from tumor-infiltrated lymph nodes. This allowed us to quantify the activation requirements of a representative polyclonal yet monospecific tumor-reactive T cell population. The results obtained from quantitative assays of intracellular Ca(2+) mobilization, TCR down-regulation, cytokine production and induction of effector cell differentiation indicate that the naturally produced Melan-A peptides are weak agonists and are clearly suboptimal for T cell activation. In contrast, optimal T cell activation was obtained by stimulation with recently defined peptide analogues. These findings provide a molecular basis for the low immunogenicity of tumor cells and suggest that patient immunization with full agonist peptide analogues may be essential for stimulation and maintenance of anti-tumor T cell responses in vivo.

Teisserenc HT, Gadola S, Cella M, Dunbar R, Exley A, Blake N, Baycal C, Lambert J, Bigliardi P, Willemsen M et al. 1999. Chronic granulomatosis lesions in patients with low surface expression of MHC class I molecules. BLOOD, 94 (10), pp. 434A-434A.

Mackensen A, Herbst B, Chen JL, Kohler G, Herr W, Cerundolo V, Lindemann A. 1999. Induction of antigen-specific immune responses in melanoma patients after vaccination with peptide-pulsed dendritic cells. BLOOD, 94 (10), pp. 216A-216A.

Moins-Teisserenc HT, Gadola SD, Cella M, Dunbar PR, Exley A, Blake N, Baykal C, Lambert J, Bigliardi P, Willemsen M et al. 1999. Association of a syndrome resembling Wegener's granulomatosis with low surface expression of HLA class-I molecules. Lancet, 354 (9190), pp. 1598-1603. | Show Abstract | Read more

BACKGROUND: Granulomatous syndromes, such as Wegener's granulomatosis, are defined according to complex criteria, but the underlying cause is rarely identified. We present evidence for a new aetiology for chronic granulomatous lesions associated with a recessive genetic defect, which is linked to the human leucocyte antigen (HLA) locus. METHODS: Five adults with necrotising granulomatous lesions in the upper respiratory tract and skin, associated with recurrent bacterial respiratory infections and skin vasculitis, were identified. A diagnosis of Wegener's granulomatosis was considered in all of them, but abandoned because of an incompatible disease course and resistance to immunosuppressive treatments. Peripheral-blood samples were taken and analysed by immunohistochemistry and fluorescent-activated-cell-sorter analysis. Since all five patients were homozygous for the HLA locus, we looked for genetic defects located within the HLA-locus with PCR and restriction fragment length polymorphism. FINDINGS: A severe decrease in cell-surface expression of HLA class-I molecule was seen in all patients. Defective expression of the transporter associated with antigen presentation (TAP) genes was responsible for the HLA class-I down-regulation, and in two patients we identified a mutation in the TAP2 gene responsible for the defective expression of the TAP complex. We showed the presence of autoreactive natural killer (NK) cells and gammadelta T lymphocytes in the peripheral blood cells of two patients. Correction of the genetic defect in vitro restored normal expression of HLA class-I molecules and prevented self-reactivity in the patients' cells. Histology of granulomatous lesions showed the presence of a large proportion of activated NK cells. INTERPRETATION: Our findings define the cause and pathogenesis of a new syndrome that affects patients with a defective surface expression of HLA class-I molecules. The syndrome resembles Wegener's granulomatosis both clinically and histologically. Patients have chronic necrotising granulomatous lesions in the upper respiratory tract and skin, recurrent infections of the respiratory tract, and skin vasculitis. A predominant NK population within the granulomatous lesions suggests that the pathophysiology of the skin lesions may relate to the inability of HLA class-I molecules to turn off NK cell responses. Accurate genetic analysis of a defined syndrome can provide a better understanding of the cause and pathogenesis of a disease.

Whelan JA, Dunbar PR, Price DA, Purbhoo MA, Lechner F, Ogg GS, Griffiths G, Phillips RE, Cerundolo V, Sewell AK. 1999. Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent. J Immunol, 163 (8), pp. 4342-4348. | Show Abstract

Tetrameric peptide-MHC class I complexes ("tetramers") are proving invaluable as reagents for characterizing immune responses involving CTLs. However, because the TCR can exhibit a degree of promiscuity for binding peptide-MHC class I ligands, there is potential for cross-reactivity. Recent reports showing that the TCR/peptide-MHC interaction is dramatically dependent upon temperature led us to investigate the effects of incubation temperature on tetramer staining. We find that tetramers rapidly stain CTLs with high intensity at 37 degrees C. We examine the fine specificity of tetramer staining using a well-characterized set of natural epitope variants. Peptide variants that elicit little or no functional cellular response from CTLs can stain these cells at 4 degrees C but not at 37 degrees C when incorporated into tetramers. These results suggest that some studies reporting tetramer incubations at 4 degrees C could detect cross-reactive populations of CTLs with minimal avidity for the tetramer peptide, especially in the tetramer-low population. For identifying specific CTLs among polyclonal cell populations such as PBLs, incubation with tetramers at 37 degrees C improves the staining intensity of specific CTLs, resulting in improved separation of tetramer-high CD8+ cells. Confocal microscopy reveals that tetramers incubated at 37 degrees C can be rapidly internalized by specific CTLs into vesicles that overlap with the early endocytic compartment. This TCR-specific internalization suggests that coupling of tetramers or analogues with toxins, which are activated only after receptor internalization, may create immunotoxins capable of killing CTLs of single specificities.

Cerundolo V. 1999. T cells work together to fight cancer. Curr Biol, 9 (18), pp. R695-R697. | Show Abstract

Recent studies have identified new melanoma antigens that are recognised by CD4(+) T cells. Analysis of tumour-specific CD4(+) T-cell responses may lead to the development of optimal anti-cancer vaccines that can induce an orchestrated effort of tumour-specific CD4(+) and CD8(+) T cells in the fight against cancer.

Speiser DE, Pittet MJ, Valmori D, Dunbar R, Rimoldi D, Liénard D, MacDonald HR, Cerottini JC, Cerundolo V, Romero P. 1999. In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J Exp Med, 190 (6), pp. 775-782. | Show Abstract | Read more

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.

Anichini A, Molla A, Mortarini R, Tragni G, Bersani I, Di Nicola M, Gianni AM, Pilotti S, Dunbar R, Cerundolo V, Parmiani G. 1999. An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J Exp Med, 190 (5), pp. 651-667. | Show Abstract | Read more

It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

Pittet MJ, Valmori D, Dunbar PR, Speiser DE, Liénard D, Lejeune F, Fleischhauer K, Cerundolo V, Cerottini JC, Romero P. 1999. High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J Exp Med, 190 (5), pp. 705-715. | Show Abstract | Read more

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Valmori D, Pittet MJ, Vonarbourg C, Rimoldi D, Liénard D, Speiser D, Dunbar R, Cerundolo V, Cerottini JC, Romero P. 1999. Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376. Cancer Res, 59 (16), pp. 4050-4055. | Show Abstract

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.

Gileadi U, Gallimore A, Van der Bruggen P, Cerundolo V. 1999. Effect of epitope flanking residues on the presentation of N-terminal cytotoxic T lymphocyte epitopes. Eur J Immunol, 29 (7), pp. 2213-2222. | Show Abstract | Read more

We here demonstrate that placing two distinct influenza virus nucleoprotein epitopes at the N terminus of a cytosolic protein selectively blocks their presentation to specific cytotoxic T lymphocytes. The block is a cytosolic phenomenon, which can be overcome by distancing the epitope from the protein N terminus by two or more amino acids. Shortening the protein's C terminus fails to relieve the antigen presentation block. These results demonstrate that events at the N terminus of the target protein, rather than at its C terminus, are responsible for the lack of presentation of N-terminal epitopes. We also show that lack of presentation of N terminal epitopes is associated with a modification of the target protein which affects its electrophoretic mobility and isoelectric focusing point. This modification can be prevented by mutating the epitope's N-terminal flanking sequence, which results in an efficient presentation of the N-terminal epitope. Lack of presentation of the N-terminal epitopes results in a reduced ability of influenza-primed mice to clear acute infection with vaccinia virus encoding an N-terminal nucleoprotein epitope. Our results demonstrate that presentation of epitopes localized at the N terminus of cytosolic proteins can be modulated by events occurring at early stages of antigen processing.

Dhodapkar MV, Steinman RM, Sapp M, Desai H, Fossella C, Krasovsky J, Donahoe SM, Dunbar PR, Cerundolo V, Nixon DF, Bhardwaj N. 1999. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. J Clin Invest, 104 (2), pp. 173-180. | Show Abstract | Read more

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Noakes KL, Teisserenc HT, Lord JM, Dunbar PR, Cerundolo V, Roberts LM. 1999. Exploiting retrograde transport of Shiga-like toxin 1 for the delivery of exogenous antigens into the MHC class I presentation pathway. FEBS Lett, 453 (1-2), pp. 95-99. | Show Abstract | Read more

Shiga-like toxin 1 (SLT) from Escherichia coli O157:H7 enters mammalian cells by endocytosis from the cell surface to the endoplasmic reticulum before translocating into the cytosol. Here, SLT was engineered at its N- or C-terminus to carry a peptide derived from influenza virus Matrix protein for delivery to major histocompatibility complex (MHC) class I molecules. We show that SLT N-Ma was capable of sensitising cells for lysis by appropriate cytotoxic T-lymphocytes whilst no killing of SLT-resistant cells was observed. Our results demonstrate that peptide was liberated intracellularly and that retrograde transport of a disarmed cytotoxic protein can intersect the MHC class 1 presentation pathway.

Dunbar PR, Chen JL, Chao D, Rust N, Teisserenc H, Ogg GS, Romero P, Weynants P, Cerundolo V. 1999. Cutting edge: rapid cloning of tumor-specific CTL suitable for adoptive immunotherapy of melanoma. J Immunol, 162 (12), pp. 6959-6962. | Show Abstract

Adoptive immunotherapy using CTL has provided some clinical benefit to patients with metastatic melanoma. Use of cloned CTL of known specificity might improve clinical effect, but technical difficulties have limited exploration of this possibility. We have used fluorescence-driven cell sorting to clone tumor-specific CTL after staining with tetrameric MHC class I/peptide complexes. CTL specific for the melanoma Ags melan-A, tyrosinase, and MAGE3 were cloned from the peripheral blood, tumor-infiltrated lymph nodes, and skin metastases of five patients. Clones were isolated and characterized in as little as 6 weeks, much faster than is possible with previous techniques. We show that these CTL clones express markers compatible with immunotherapeutic use in melanoma, including the cutaneous lymphocyte Ag, which is associated with homing to skin.

Sewell AK, Price DA, Teisserenc H, Booth BL, Gileadi U, Flavin FM, Trowsdale J, Phillips RE, Cerundolo V. 1999. IFN-gamma exposes a cryptic cytotoxic T lymphocyte epitope in HIV-1 reverse transcriptase. J Immunol, 162 (12), pp. 7075-7079. | Show Abstract

The proteasome, an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells, is responsible for the degradation of most cellular proteins and is believed to be the main source of MHC class I-restricted antigenic peptides for presentation to CTL. Inhibition of the proteasome by lactacystin or various peptide aldehydes can result in defective Ag presentation, and the pivotal role of the proteasome in Ag processing has become generally accepted. However, recent reports have challenged this observation. Here we examine the processing requirements of two HLA A*0201-restricted epitopes from HIV-1 reverse transcriptase and find that they are produced by different degradation pathways. Presentation of the C-terminal ILKEPVHGV epitope is impaired in ME275 melanoma cells by treatment with lactacystin, and is independent of expression of the IFN-gamma-inducible proteasome beta subunits LMP2 and LMP7. In contrast, both lactacystin treatment and expression of LMP7 induce the presentation of the N-terminal VIYQYMDDL epitope. Consistent with these observations we show that up-regulation of LMP7 by IFN-gamma enhances presentation of the VIYQYMDDL epitope. Hence interplay between constitutive and IFN-gamma-inducible beta-subunits of the proteasome can qualitatively influence Ag presentation. These observations may have relevance to the patterns of immunodominance during the natural course of viral infection.

Speiser DE, Valmori D, Rimoldi D, Pittet MJ, Liénard D, Cerundolo V, MacDonald HR, Cerottini JC, Romero P. 1999. CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. Eur J Immunol, 29 (6), pp. 1990-1999. | Show Abstract | Read more

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.

Bachmann MF, Gallimore A, Linkert S, Cerundolo V, Lanzavecchia A, Kopf M, Viola A. 1999. Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells. J Exp Med, 189 (10), pp. 1521-1530. | Show Abstract | Read more

The question of whether enhanced memory T cell responses are simply due to an increased frequency of specific cells or also to an improved response at the single cell level is widely debated. In this study, we analyzed T cell receptor (TCR) transgenic memory T cells and bona fide memory T cells isolated from virally infected normal mice using the tetramer technology. We found that memory T cells are qualitatively different from naive T cells due to a developmentally regulated rearrangement of the topology of the signaling machinery. In naive cytotoxic T cells, only a few CD8 molecules are associated with Lck and the kinase is homogeneously distributed inside the cell. However, in vivo priming of naive T cells induces the targeting of Lck to the CD8 coreceptor in the cell membrane and the consequent organization of a more efficient TCR signaling machinery in effector and memory cells.

Valmori D, Pittet MJ, Rimoldi D, Liénard D, Dunbar R, Cerundolo V, Lejeune F, Cerottini JC, Romero P. 1999. An antigen-targeted approach to adoptive transfer therapy of cancer. Cancer Res, 59 (9), pp. 2167-2173. | Show Abstract

Previous attempts to treat human malignancies by adoptive transfer of tumor-specific CTLs have been limited by the difficulty of isolating T cells of defined antigen specificity. The recent development of MHC class I/antigenic peptide tetrameric complexes that allow direct identification of antigen-specific T cells has opened new possibilities for the isolation and in vitro expansion of tumor-specific T cells. In the present study, we have derived polyclonal monospecific cell lines from circulating Melan-A-specific CTL precursors of HLA-A*0201+ melanoma patients by combining stimulation with recently identified peptide analogues of the immunodominant epitope from the melanoma-associated antigen Melan-A with staining with fluorescent HLA-A*0201/Melan-A peptide tetramers. In vitro expansion of antigen-specific CD8+ T cells was monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. This analysis revealed that Melan-A 26-35 peptide analogues were much more efficient than the parental peptides in stimulating a rapid in vitro expansion of antigen-specific CD8+ T cells. These cells were then isolated by tetramer-guided cell sorting and subsequently expanded in vitro by mitogen stimulation. The resulting polyclonal but monospecific CTLs fully cross-recognized the parental peptides and were able to efficiently lyse Melan-A-expressing tumor cells. Altogether, these results pave the way to a molecularly defined approach to antigen-specific adoptive transfer therapy of cancer.

Valmori D, Gileadi U, Servis C, Dunbar PR, Cerottini JC, Romero P, Cerundolo V, Lévy F. 1999. Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3. J Exp Med, 189 (6), pp. 895-906. | Show Abstract | Read more

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

Collins EJ, Booth BL, Cerundolo V. 1999. Extensive alanine substitutions increase binding affinity of an influenza nucleoprotein peptide to HLA-Aw68 and do not abrogate peptide-specific CTL recognition. J Immunol, 162 (1), pp. 331-337. | Show Abstract

Class I MHC molecules bind peptides in the endoplasmic reticulum and present them at the cell surface to circulating CD8+ T cells for analysis. We have examined binding of peptides and stabilization of HLA-Aw68 class I molecules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR). We have demonstrated that insertion of increasing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t 1/2 of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently. Peptide variants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line. Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residues exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some of these polyalanine peptides are recognized by T cell lines generated against NP91-99. Analysis of the peptide sequences show that the stabilization effects are not due to the hydrophobicity of the peptide. These data suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable contacts between the peptide ligand and class I MHC-binding cleft.

Romero P, Dunbar PR, Valmori D, Pittet M, Ogg GS, Rimoldi D, Chen JL, Liénard D, Cerottini JC, Cerundolo V. 1998. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes. J Exp Med, 188 (9), pp. 1641-1650. | Show Abstract | Read more

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Young NT, Rust NA, Dallman MJ, Cerundolo V, Morris PJ, Welsh KI. 1998. Independent contributions of HLA epitopes and killer inhibitory receptor expression to the functional alloreactive specificity of natural killer cells. Hum Immunol, 59 (11), pp. 700-712. | Show Abstract | Read more

Human NK cells express receptors (KIR) which inhibit lysis through binding to HLA class I on target cells. KIR expression in different individuals has not been intensively investigated and it is not known how the KIR repertoire relates to HLA type or influences the overall activity of NK populations. This may be important in the response of NK cells to HLA-mismatched organ transplants since the ligands for KIR are supertypic epitopes shared between certain HLA alleles. We studied the effect of matching for HLA on the cytotoxicity of NK cells from individuals homozygous or heterozygous for relevant HLA class I epitopes and correlated this with KIR expression and genotype. Considerable variation in the KIR repertoire of different donors was evident, including functional KIR expressed in the absence of specific HLA ligands. We confirmed the predominant influence of HLA-C in a hierarchy of inhibitory effects mediated by HLA class I loci. In certain individuals, inhibition patterns are more complicated and may be due to the relative expression of the CD94/NKG2 receptors. Our study reveals the separate contributions of HLA and KIR molecules to NK cell alloreactivity and provides a basis for consideration of the functional diversity of KIR genes in transplantation.

André P, Groettrup M, Klenerman P, de Giuli R, Booth BL, Cerundolo V, Bonneville M, Jotereau F, Zinkernagel RM, Lotteau V. 1998. An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc Natl Acad Sci U S A, 95 (22), pp. 13120-13124. | Show Abstract | Read more

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Young NT, Mulder A, Cerundolo V, Claas FH, Welsh KI. 1998. Expression of HLA class I antigens in transporter associated with antigen processing (TAP)-deficient mutant cell lines. Tissue Antigens, 52 (4), pp. 368-373. | Show Abstract | Read more

Cells lacking expression of the transporter associated with antigen processing (TAP) are deficient in surface HLA class I, yet express reduced levels of HLA-A2 antigen through TAP-independent processing pathways. We have analysed the expression of HLA-A, -B and -C antigens on the 721.174 and T2 TAP-deficient mutant cell lines using a panel of monoclonal antibodies specific for the HLA antigens encoded by the genotype of these cells. Our study has shown the constitutive expression of HLA-Cw1 molecules on the cell surface of both T2 and 721.174 cells and has confirmed that HLA-A2 and HLA-B51 are expressed at low levels. Transfection of 721.174 cells with cDNAs encoding TAP1 and TAP2 proteins did not fully restore HLA class I antigen expression on these cells, which appeared to be mainly due to a deficiency in expression of the HLA-B51-associated Bw4 epitope. This suggests that additional antigen-processing genes may be required for optimal generation of HLA-B-binding peptides. Our results indicate that TAP-independent pathways of antigen-processing provide peptides for functional expression of all three classical HLA class I molecules.

Ogg GS, Rod Dunbar P, Romero P, Chen JL, Cerundolo V. 1998. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. J Exp Med, 188 (6), pp. 1203-1208. | Show Abstract | Read more

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2-MelanA tetramer+ CTLs) in seven of nine HLA-A*0201-positive individuals with vitiligo. Isolated A2-MelanA tetramer+ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2-MelanA tetramer+ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2-MelanA tetramer+ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo.

Colonna M, Samaridis J, Cella M, Angman L, Allen RL, O'Callaghan CA, Dunbar R, Ogg GS, Cerundolo V, Rolink A. 1998. Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I molecules. J Immunol, 160 (7), pp. 3096-3100. | Show Abstract

Leukocyte activation can be negatively regulated by inhibitory receptors specific for MHC class I molecules. While one inhibitory receptor, Ig-like transcript 2 (ILT2), is expressed by all lymphoid and myelomonocytic cell types, other receptors display a more selective tissue distribution. Here we characterize an inhibitory receptor, termed ILT4, which is selectively expressed in monocytes, macrophages, and dendritic cells (DCs), binds classical class I molecules and the nonclassical class I molecules HLA-G, and transduces negative signals that can inhibit early signaling events triggered by stimulatory receptors. ILT4 may control inflammatory responses and cytotoxicity mediated by myelomonocytic cells and may modulate their Ag-presenting functions, focusing immune responses to microbial challenges and avoiding autoreactivity.

Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V et al. 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science, 279 (5359), pp. 2103-2106. | Show Abstract | Read more

Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.

Dunbar PR, Ogg GS, Chen J, Rust N, van der Bruggen P, Cerundolo V. 1998. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood. Curr Biol, 8 (7), pp. 413-416. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infections and certain tumours, but characterising specific CTL responses has always been technically limited. Fluorogenic 'tetramers' of major histocompatibility complex (MHC) class I complexes have been exploited recently to quantify the massive expansion of specific CTLs in human immunodeficiency virus (HIV) infection [1]. Here, we use MHC class I complex tetramers to isolate low-frequency antigen-specific CTLs directly from human peripheral blood, allowing the simultaneous phenotypic and functional characterisation and cloning of these CTLs. We synthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2. 1-restricted CTL clones recognising the influenza matrix protein peptide 58-66, matrix 58-66 [2]. This tetramer stained between 1 in 1,500 and 1 in 58,000 peripheral blood mononuclear cells (PBMCs) from HLA-A2.1+ individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the cells could be directly sorted into enzyme-linked immunospot (ELISpot) plates, where they released interferon-gamma (IFN-gamma) within 1 day of antigen exposure. The same population was cloned by FACS, and the specificity of several expanded clones was confirmed. Cloning was greatly simplified and accelerated compared with standard protocols, and was highly efficient. We also used tetramer-based sorting to enrich melanoma-specific CTLs derived from a tumour-infiltrated lymph node. Direct cloning of specific CTLs from peripheral blood can provide important information about immunological memory, CTL responses against tumour antigens and CTL proliferation and function, and opens up new possibilities for generating CTLs for adoptive immunotherapy.

Cerundolo V, Booth B, Gileadi U, Sewell A, Phillips R, Trowsdale J, Teisserenc H. 1998. Role of the proteasome and non proteasomal proteases in the generation of CTL epitopes. FASEB JOURNAL, 12 (5), pp. A1087-A1087. | Show Abstract

We have previously shown that cells lacking the MHC encoded proteasome subunits LMP2 and LMP7 have a severe defect in the generation of the HLA-A2 restricted influenza Matrix epitope 58-66. We have now extended this observation to other viral epitopes and demonstrated that the antigen presentation block can be restored by transfecting LMP7. We also show that the LMP7 dependent presentation of the Matrix epitope 58-66 can be overcome by expressing fragments of the influenza Matrix, up to 100 amino acid long These results demonstrate that a single proteasome subunit is critical for the generation of defined CTL epitopes and highlight the possibility that either the length or folding of cytosolic proteins may influence the processing pathway leading to the generation of CTL epitopes. Consistent with the latter possibility we demonstrate that processing of a rapidly degraded CTL target protein is redirected at 42°C from the proteasomes to non-proteasomal cytosolic proteases. Heat shock dependent protein degradation has a profound effect on the repertoire of MHC class I bound antigenic peptides, as cells incubated at 42°C lose the ability of generating a defined CTL epitope but presentation of another epitope is significantly enhanced. The results demonstrate that antigenic peptides can be generated by different processing pathways and highlight the effect of heat shock on processing and presentation of CTL proteins.

Springer S, Doring K, Skipper JC, Townsend AR, Cerundolo V. 1998. Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding. Biochemistry, 37 (9), pp. 3001-3012. | Show Abstract | Read more

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

Braud VM, McMichael AJ, Cerundolo V. 1998. Differential processing of influenza nucleoprotein in human and mouse cells. Eur J Immunol, 28 (2), pp. 625-635. | Show Abstract | Read more

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265-273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265-273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.

Teisserenc H, Schmitt W, Blake N, Dunbar R, Gadola S, Gross WL, Exley A, Cerundolo V. 1997. A case of primary immunodeficiency due to a defect of the major histocompatibility gene complex class I processing and presentation pathway. Immunol Lett, 57 (1-3), pp. 183-187. | Show Abstract | Read more

INTRODUCTION: We report a case of primary immunodeficiency due to a defect of the TAP transporter, an heterodimeric complex which controls the expression of HLA class I molecule by delivering peptides from the cytosol into the lumen of the endoplasmic reticulum. Since childhood, the 36 year old female suffered from recurrent sinusitis/bronchitis. She later developed bronchiectasis and destructive nasal epitheloid granulomata in conjunction with a generalized vasculitic syndrome that did not improve upon immunosuppression and antibiotics. METHODS: The class I monomorphic W6/32 was used for cell surface staining and immunoprecipitation of MHC class I molecules. Peptide transport assay was carried out in semi-permeabilized cells with iodinated peptides. Antigen presentation experiments were performed using chromium 51 labelled patient B cell line and EBV specific CTL. TAP1 and TAP2 specific antibodies were used for Western blotting and immunoprecipitation of the TAP complex. RESULTS AND CONCLUSIONS: A severe reduction of MHC class I molecules at the cell surface of the B-cell lines was observed, whereas MHC class II expression was not altered. Isoelectric focusing of metabolically labelled MHC class I molecules revealed that class I heavy chains remain unsialylated, consistent with a block of TAP dependent peptide translocation. These conclusions were confirmed by further experiments showing that peptide translocation was completely abolished. We also demonstrated that presentation of viral antigens through endogenous class I molecules was severely impaired. Immunoprecipitation and Western blotting of TAP1/2 complex showed that TAP2 was not detectable. Further, experiments are in progress to identify the site of the mutation.

Cerundolo V, Benham A, Braud V, Mukherjee S, Gould K, Macino B, Neefjes J, Townsend A. 1997. The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells. Eur J Immunol, 27 (1), pp. 336-341. | Show Abstract | Read more

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza-specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome-dependent degradation plays an important role in the cytosolic generation of CTL epitopes.

Healey C, Chrisite J, Lai M, Cerundolo V, Rosenberg W. 1996. Mutations in a putative hepatitis B virus HLA-A1 cytotoxic T lymphocyte epitope. Affect binding and correlate with disease severity. GASTROENTEROLOGY, 110 (4), pp. A1209-A1209.

Tomiyama H, Takamiya Y, Hill AB, Cerundolo V, Kelly A, Egawa K, Trowsdale J, Takiguchi M. 1995. Isolation of a human allo-peptide presented by HLA-B51 molecules. Int Immunol, 7 (6), pp. 905-909. | Show Abstract | Read more

Recent studies have demonstrated directly that alloreactive mouse CTL recognize peptides presented by MHC class I molecules. However, there is no direct evidence that human alloreactive CTL recognize peptides presented by HLA class I molecules. We have isolated an HLA-B51 alloreactive CTL clone, 2B3, that did not kill the TAP defective cell lines T2 and .174, whereas it killed the TAP-positive cell line T1 and .174 cells transfected with TAP genes. These findings suggested that this clone recognizes a TAP-dependent allo-peptide. We attempted to isolate the human allo-peptide recognized by the 2B3 clone from HLA-B51 molecules. A naturally occurring HLA-B*5101 binding peptide isolated from T1 cells was recognized by the 2B3 clone. The peptide was also isolated from HLA-B*5101 molecules purified from C1R-B*5101 cells. In the present study, we directly demonstrated that a human alloreactive CTL clone recognizes peptide presented by HLA class I molecules.

Elliott T, Willis A, Cerundolo V, Townsend A. 1995. Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum. J Exp Med, 181 (4), pp. 1481-1491. | Show Abstract | Read more

We have introduced long precursor peptides directly into the endoplasmic reticulum (ER) of a mutant cell line (T2-Db) that lacks the ability to transport peptides from the cytosol to the ER in a transporter associated with antigen processing (TAP) dependent way. This was done by expressing various influenza A-derived peptides containing the naturally processed epitope ASNENMDAM (366-374) preceded by the influenza hemagglutinin ER translocation sequence. Peptides derived from these minigenes that became associated with Db were isolated and identified by combined reversed phase liquid chromatography and detection by cytotoxic T lymphocytes. Our results establish that NH2-terminal extensions of at least 40 residues can be trimmed from peptides entering the ER, but that proteolysis of larger proteins may be limited.

Cerundolo V, Kelly A, Elliott T, Trowsdale J, Townsend A. 1995. Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport. Eur J Immunol, 25 (2), pp. 554-562. | Show Abstract | Read more

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.

Elvin J, Potter C, Elliott T, Cerundolo V, Townsend A. 1993. A method to quantify binding of unlabeled peptides to class I MHC molecules and detect their allele specificity. J Immunol Methods, 158 (2), pp. 161-171. | Show Abstract | Read more

A general method has been developed for measuring the stabilization of class I MHC molecules in extracts of the mutant cell lines .174/T2 and RMA-S. 35S-Met-labeled class I molecules which have been stabilized by peptides in vitro are immunoprecipitated with conformation dependent monoclonal antibodies and electrophoresed on polyacrylamide gels. The heavy and light chains are excised from the dried gel and quantified on a flat bed scintillation counter. The stabilizing effect of peptides on class I molecules in vitro correlates well with peptide binding measured by direct methods and can be therefore used to assess peptide binding affinity. We show that a peptide from HIV-1 gag (which has a high affinity for Db) is a CTL epitope restricted through Db, and also use the assay to analyse the effects of amino acid substitution on peptide affinity. In addition, the effect of a given peptide on a class I molecule within a mixture of human class I molecules can be distinguished by immunoprecipitation with the monomorphic antibody W6/32 and separation by 1-D isoelectric focussing. The technique therefore requires neither labeled peptide ligands nor allele-specific antibodies. It can be used to identify the peptide ligand of any human class I molecule, and gives a measure of peptide binding affinity. The technique should be of value in identifying epitopes recognized by CTL since we have found that these tend to bind with the highest affinities.

Cerundolo V. 1993. Antigen processing and presentation. The role of the endoplasmic reticulum. Subcell Biochem, 21 pp. 209-228.

Elliott T, Cerundolo V, Townsend A. 1992. Short peptides assist the folding of free class I heavy chains in solution. Eur J Immunol, 22 (12), pp. 3121-3125. | Show Abstract | Read more

Previous experiments have shown that short peptides coresponding to naturally processed epitopes of viral antigens can induce a conformational change in the class I heavy chain (HC) to which they bind in the fully assembled molecule. Here, we present evidence that the mechanism for this conformational change may involve binding of peptide to a partially unfolded form of free HC, followed by its subsequent folding. These results may be important for understanding the way in which class I molecules are assembled in vivo, and how certain epitopes are selected for presentation to T cells.

Cerundolo V, Elliott T, Elvin J, Bastin J, Townsend A. 1992. Association of the human invariant chain with H-2 Db class I molecules. Eur J Immunol, 22 (9), pp. 2243-2248. | Show Abstract | Read more

We describe two proteins of 24 kDa and 33 kDa (p24 and p33) which associate with H-2 Kb and H-2 Db molecules, respectively, in human cells transfected with H-2 Kb and H-2 Db genes. This association is particularly clear in the mutant cell line T2, in which association of endogenous peptide with newly synthesized class I molecules may not occur (V. Cerundolo et al., Nature 1990. 345: 449). We show that p33 is the 33-kDa form of the human invariant chain which is resident in the endoplasmic reticulum of T2 cells (P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 1989. LIV:309). The stability of the invariant chain H-2 Db complex is critically dependent upon occupation of the class I binding site by peptide ligand. In the absence of peptide, the complex is stable at 4 degrees C whereas following exposure to peptide, the invariant chain dissociates rapidly from H-2 Db molecules (half-life of 30 min at 4 degrees C). Although the interaction between the human invariant chain and murine H-2 Db is unlikely to have any functional significance, the peptide-induced dissociation of the invariant chain is consistent with a conformational change in H-2 Db on peptide binding.

Elliott T, Elvin J, Cerundolo V, Allen H, Townsend A. 1992. Structural requirements for the peptide-induced conformational change of free major histocompatibility complex class I heavy chains. Eur J Immunol, 22 (8), pp. 2085-2091. | Show Abstract | Read more

In an attempt to define the structural features of peptides which are important for inducing the folding of free class I heavy chains in the absence of beta 2-microglobulin, and to determine whether they are the same as those required to form stable major histocompatibility complex (MHC): peptide adducts, we have used a panel of peptides related to the Db-binding nonamer ASNENMDAM (influenza nucleoprotein residues 366-374) with altered primary structures, and a number of other peptides which have the Db-binding "motif". In this way, we have shown that in addition to the "anchor" residues which define this motif, the alpha amino and carboxyl groups at the N and C termini also play a major role in both inducing the conformational change in free heavy chain (HC) and formation of a stable Db:peptide complex. We also show that the importance of the key residues is affected by the primary sequence "context" in which they appear. In addition, we have extended our original finding that naturally processed epitopes induce a conformational change in free HC to the H2Kb HC, and show that the effect does not require the presence of the class I alpha 3 domain.

van Binnendijk RS, van Baalen CA, Poelen MC, de Vries P, Boes J, Cerundolo V, Osterhaus AD, UytdeHaag FG. 1992. Measles virus transmembrane fusion protein synthesized de novo or presented in immunostimulating complexes is endogenously processed for HLA class I- and class II-restricted cytotoxic T cell recognition. J Exp Med, 176 (1), pp. 119-128. | Show Abstract | Read more

The routes used by antigen-presenting cells (APC) to convert the transmembrane fusion glycoprotein (F) of measles virus (MV) to HLA class I and class II presentable peptides have been examined, using cloned cytotoxic T lymphocytes in functional assays. Presentation by Epstein-Barr virus-transformed B lymphoblastoid cell lines was achieved using live virus, ultraviolet light-inactivated virus, and purified MV-F delivered either as such or incorporated in immunostimulating complexes (MV-F-ISCOM). Only live virus and MV-F-ISCOM allow presentation by class I molecules, while all antigen preparations permit class II-restricted presentation. We observe presentation of MV-F from live virus and as MV-F-ISCOM by class II molecules in a fashion that is not perturbed by chloroquine. Our studies visualize novel presentation pathways of type I transmembrane proteins.

Spies T, Cerundolo V, Colonna M, Cresswell P, Townsend A, DeMars R. 1992. Presentation of viral antigen by MHC class I molecules is dependent on a putative peptide transporter heterodimer. Nature, 355 (6361), pp. 644-646. | Show Abstract | Read more

Major histocompatibility complex (MHC) class I molecules present peptides derived from the endogenous protein pool to cytotoxic T lymphocytes, which can thus recognize intracellular antigen. This pathway may depend on a transporter (PSF1) to mediate entry of the cytosolic peptides into a pre-Golgi compartment where they bind to class I heavy chains and promote their stable assembly with beta 2-microglobulin. There is, however, only indirect support for this function of PSF1. Here we show that PSF1 is necessary for the efficient assembly of class I molecules and enables them to present a peptide epitope derived from endogenously synthesized viral antigen. Immunochemical and genetic data demonstrate that the PSF1 polypeptide is associated with a complementary transporter chain, which is polymorphic and is encoded by the PSF2 gene, which is closely linked to PSF1.

Zanovello P, Rosato A, Bronte V, Mandruzzato S, Cerundolo V, Collavo D. 1992. Antitumour efficacy of lymphokine-activated killer cells loaded with ricin against experimentally induced lung metastases. Cancer Immunol Immunother, 35 (1), pp. 27-32. | Show Abstract | Read more

Adoptive transfer of tumour-specific T lymphocytes loaded with ricin into tumour-bearing mice exerts a transient therapeutic effect against locally induced tumours [Cerundolo et al. (1987) Br J Cancer 55: 413]. As transferred cells preferentially locate in the lung, we studied the therapeutic effect of ricin-loaded, lymphokine-activated killer (LAK) cells on lung metastases induced by M4 or B16-F1 (F1) tumour cell injection. In vitro studies demonstrated that ricin-treated LAK cells were about 100-fold more efficient than untreated LAK cells in inhibiting growth of the ricin-sensitive M4 tumour cell line. This effect was most likely due to the released ricin, as treated and untreated LAK cells inhibited the relatively toxin-resistant F1 cell line to the same extent. Ricin treatment did not alter the tissue distribution of intravenously (i.v.) injected LAK cells, which selectively localized in the lung early after inoculation, whether or not metastases were present. Adoptive transfer experiments showed that ricin-treated LAK cells were significantly more efficient than untreated LAK cells in inhibiting M4- but not F1-induced lung metastases. These results indicate that LAK cells are able to deliver a therapeutic concentration of antineoplastic compounds directly to the lung.

Elvin J, Cerundolo V, Elliott T, Townsend A. 1991. A quantitative assay of peptide-dependent class I assembly. Eur J Immunol, 21 (9), pp. 2025-2031. | Show Abstract | Read more

We have developed a quantitative assay for the measurement of class I assembly induced by peptide. We have applied this assay to H-2Db, Kb and HLA-A2.1 with a panel of 49 overlapping peptides derived from HIV-1 gag protein. We find that the effects of peptide on assembly form a continuous distribution. By defining positives as those that increase the concentration of folded heavy chains more than three standard deviations from the control we show that 7/48 bind A2.1, 11/49 bind Db and 7/47 bind Kb. The assembly assay contrasts with solid-phase assays in being more discriminating (fewer peptides binding any given class I molecule), and showing less overlap in the patterns of peptides bound by the three class I molecules.

Cerundolo V, Elliott T, Elvin J, Bastin J, Rammensee HG, Townsend A. 1991. The binding affinity and dissociation rates of peptides for class I major histocompatibility complex molecules. Eur J Immunol, 21 (9), pp. 2069-2075. | Show Abstract | Read more

Peptides of various lengths derived from the influenza nucleoprotein (NP) bind to H-2Db class I molecules with affinities at 4 degrees C between approximately 3 x 10(5)- approximately 3 x 10(7) M-1. The peptide with the highest affinity corresponds to the sequence of nine amino acids (NP366-374) recently isolated from cells infected with influenza. This peptide forms stable complexes with half-lives greater than 110 h at 4 degrees C, 39 h at 22 degrees C and 3 h at 37 degrees C. Small increases in length of the peptide greatly reduce the stability of the complex (t1/2 approximately 1-10 h at 4 degrees C). These results may explain the homogeneous length of peptides isolated from class I molecules formed in vivo, and suggest that class I and II may differ in their dependence on the length of peptides for the formation of stable complexes.

Elliott T, Cerundolo V, Elvin J, Townsend A. 1991. Peptide-induced conformational change of the class I heavy chain. Nature, 351 (6325), pp. 402-406. | Show Abstract | Read more

There is evidence that peptide ligands take part in the assembly of class I molecules. In particular, addition of peptides to extracts of the mutant cells RMA-S and .174/T2, in which stable assembly of class I does not occur, results in a conformational change in the class I heavy chain and stable association of the heavy chain with beta 2-microglobulin (beta 2m). Thus specific peptides may stabilize or induce a conformational change in the class I heavy chain that results in a rise in the binding affinity of the heavy chain for beta 2m (Fig. 1a). Here we show that peptides have two cooperative roles in class I assembly. Specific short peptides (9-10 amino acids) can induce folding of the heavy chain in the absence of beta 2m. Both short (nine amino acids) and longer sequences (15 amino acids) can stabilize performed low-affinity complexes of heavy chain and beta 2m. To alter the conformation of free heavy chains, the peptides must be exactly the correct size, and they are found to correspond to the sequences isolated from infected cells. This property may therefore be the basis for selection of epitopes presented in vivo.

Cerundolo V, Tse AG, Salter RD, Parham P, Townsend A. 1991. CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1. Proc Biol Sci, 244 (1310), pp. 169-177. | Show Abstract | Read more

The crystal structure of the HLA-Aw68.1 antigen binding site revealed a negatively charged pocket centred on aspartic acid 74 (Garrett et al. 1989). Access to this '74 pocket' is blocked in HLA-Aw68.2 and HLA-Aw69 by two substitutions at positions 97 and 116. This key feature suggests that the Aw68.1-peptide-specific interactions may involve salt bridges between oppositely charged residues. In this paper, the influenza epitope recognized by virus-specific HLA-Aw68.1-restricted cytotoxic T lymphocytes (CTL) has been defined in vitro with a synthetic peptide corresponding to residues 89-101 of the nucleoprotein (NP). Amino acid substitutions of the peptide NP 89-101 showed that the arginine at position 99 is an anchor point of the peptide within the Aw68.1 antigen binding site. Consistent with this we find that neither HLA-Aw68.2 nor HLA-Aw69 positive cells can present peptide NP 89-101 to Aw68.1-restricted CTL. Our results therefore suggest a model in which presentation of NP 89-101 by HLA-Aw68.1 is dependent upon interaction of the positively charged arginine residue at position 99 of the peptide, with the negatively charged aspartic acid in the '74 pocket' of HLA-Aw68.1. We also show that influenza-virus-specific HLA-Aw68.1-restricted CTL are CD8 independent. This result is consistent with the low affinity of HLA-Aw68.1 for CD8 (Salter et al. 1989) and reveals a unique example of CD8-independent priming of CTL by natural infection with a common pathogen in humans.

Zanovello P, Rosato A, Bronte V, Cerundolo V, Collavo D. 1991. Adoptive immunotherapy of experimental tumors using cytotoxic lymphocytes to carry and deliver toxins. Ann Ist Super Sanita, 27 (1), pp. 91-95. | Show Abstract

Adoptive transfer of specifically sensitized lymphoid cells constitutes a potential tool for specific cancer immunotherapy, however, the requirement of syngeneic or autologous specifically reactive cells has limited its use in the treatment of human cancer. In several mouse tumor models, large amounts of lymphokine activated killer (LAK) cells associated with high doses of interleukin 2 (IL-2) are able to mediate the regression of established pulmonary and hepatic metastases. Since LAK cells are more easily generated than specifically sensitized lymphocytes and show broad tumor specificity, this approach has been applied in humans to treat cancer, but the acute toxicity associated with the high doses of IL-2 administered represents an important drawback to its clinical use. The observation that lymphocytes can internalize ricin, a toxic plant protein, and then release it in an active form, capable of destroying other cells, led us to investigate the possibility of using antigen-specific cytotoxic T lymphocytes (CTL) or LAK cells as carriers of toxic substances to the tumor site. In our experimental models we observed that tumor-specific CTL or LAK cells can be used to deliver ricin into the tumor mass and cause temporary tumor growth inhibition.

Elliott TJ, Cerundolo V, Ohlen C, Ljunggren HG, Karre K, Townsend A. 1991. Antigen presentation and the association of class-I molecules. Acta Biol Hung, 42 (1-3), pp. 213-229. | Show Abstract

We have identified two mutant cell lines which are not able to present epitopes of influenza virus synthesized in the cytoplasm but can present the same epitope when exposed to it as a peptide in the extracellular medium. The cell lines also have a defect in class-I assembly, with reduced expression of assembled alpha chain: beta 2M heterodimers at their cell surface. This led to the suggestion that the two traits were the result of the same mutation and that stable assembly of class-I molecules is dependent on peptide binding. Consistent with this idea was the finding that exposure to specific peptides in the extracellular fluid promotes stable association of class-I heavy chains with beta 2M and restores expression of class-I at the cell surface. We have gone on to show that stable assembly of class-I molecules can be supported in detergent extracts of the mutant cells when specific peptides are added. Peptides stabilized a conformational change in the class-I heavy chain and association with beta 2M by binding to the complexes. This effect is apparent at peptide concentrations around 100-fold lower than required in "peptide feeding" experiments with whole cells. We have also demonstrated that the conformational change induced in heavy chain is influenced by the concentration of beta 2M, and consequently have been able to demonstrate the formation of empty class-I molecules.

Elliott T, Townsend A, Cerundolo V. 1990. Antigen presentation. Naturally processed peptides. Nature, 348 (6298), pp. 195-197. | Read more

Townsend A, Elliott T, Cerundolo V, Foster L, Barber B, Tse A. 1990. Assembly of MHC class I molecules analyzed in vitro. Cell, 62 (2), pp. 285-295. | Show Abstract | Read more

Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.

Cerundolo V, Alexander J, Anderson K, Lamb C, Cresswell P, McMichael A, Gotch F, Townsend A. 1990. Presentation of viral antigen controlled by a gene in the major histocompatibility complex. Nature, 345 (6274), pp. 449-452. | Show Abstract | Read more

We describe a mutant human cell line (LBL 721.174) that has lost a function required for presentation of intracellular viral antigens with class I molecules of the major histocompatibility complex (MHC), but retains the capacity to present defined epitopes as extracellular peptides. The cell also has a defect in the assembly and expression of class I MHC molecules, which we show can be restored by exposure of the cells to a peptide epitope. This phenotype suggests a defect in the association of intracellular antigen with class I molecules similar to that described for the murine mutant RMA-S (ref. 5), but in the present case the genetic defect can be mapped within the MHC locus on human chromosome 6.

Elliott T, Townsend A, Cerundolo V. 1990. Naturally processed peptides Nature, 348 (6298), pp. 195-197. | Read more

Zanovello P, Rosato A, Bronte V, Cerundolo V, Treves S, Di Virgilio F, Pozzan T, Biasi G, Collavo D. 1989. Interaction of lymphokine-activated killer cells with susceptible targets does not induce second messenger generation and cytolytic granule exocytosis. J Exp Med, 170 (3), pp. 665-677. | Show Abstract | Read more

CTL activation by specific targets leads to a rapid rise of inositol phosphates (InsPs) and of cytoplasmic-free Ca2+ concentration ([Ca2+]i). While these events are considered necessary to trigger granule secretion, Ca2+-independent cytolytic mechanisms have been recently proposed in addition or as an alternative to the classical Ca2+-dependent exocytosis model. We observed that lymphokine-activated killer (LAK) cells, obtained after stimulation with supraoptimal concentrations of IL-2 in short- or long-term cultures, kill susceptible targets in the absence of a [Ca2+]i rise and InsP3 formation. Moreover, LAK cell-mediated lysis was not associated with an increase in cytotoxic granule exocytosis, as evaluated by BLT-esterase release into the culture supernatant. Furthermore, using an antigen-specific CTL clone, which acquires LAK-like activity when cultured in medium containing high IL-2 doses, second messenger generation and cytolytic granule content secretion were not detected during lysis of unrelated target cells, while killing of specific targets triggered both these processes. These findings suggest that two lytic pathways may coexist in the same effector cells: a second messenger-dependent pathway involving degranulation, which is activated after TCR interaction with specific targets, and another pathway, independent of any known second messenger generation, responsible for unrelated target cell lysis.

Zanovello P, Cerundolo V, Bronte V, Giunta M, Panozzo M, Biasi G, Collavo D. 1989. Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity. Cell Immunol, 122 (2), pp. 450-460. | Show Abstract | Read more

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.

Treves S, Di Virgilio F, Cerundolo V, Zanovello P, Collavo D, Pozzan T. 1987. Calcium and inositolphosphates in the activation of T cell-mediated cytotoxicity. J Exp Med, 166 (1), pp. 33-42. | Show Abstract | Read more

Reports from a number of laboratories have shown that mAbs against the T3-Ti receptor complex cause an increase in cytosolic-free Ca2+ [( Ca2+]i) and the hydrolysis of phosphatidylinositolbisphosphate (PIP2) in CTLs. In the present report we show that activation of CTLs by their specific targets causes: (a) release of Ca2+ from intracellular stores; (b) transient formation of inositol trisphosphate (InsP3); and (c) an increased permeability to Ca2+ of CTL plasma membrane. Killing of unrelated targets could be induced by cocentrifugation of the unrelated targets with CTLs in the presence of A23187 or PMA. We conclude that: (a) activation of CTLs by specific antigens triggers the generation of the same intracellular mediators generated by stimulation of lymphocytes with anti-T3-Ti receptor antibodies and/or with polyclonal mitogens; and (b) intracellular signals that mediate the delivery of the lethal hit by CTLs are indistinguishable from those that induce cell proliferation.

Cerundolo V, Zanovello P, McIntosh D, Fabbris R, Davies AJ, Collavo D. 1987. Temporary inhibition of Moloney-murine sarcoma virus (M-MSV) induced-tumours by adoptive transfer of ricin-treated T-lymphocytes. Br J Cancer, 55 (4), pp. 413-419. | Show Abstract | Read more

The potential use of tumour-specific T-lymphocytes loaded with ricin in cell targeting experiments was investigated. Moloney-murine sarcoma virus (M-MSV)-specific T-lymphocytes, obtained in mass mixed leucocyte-tumour cell culture (MLTC) and a M-MSV-specific cytotoxic T-lymphocyte (CTL) clone, were incubated with 125I-labelled ricin in order to evaluate toxin uptake and release. The internalized ricin (4.5 X 10(-17) mol and 6.5 X 10(-17) mol per 10(2) MLTC and CTL clone cells, respectively) was released rapidly during the first 30 min following treatment, and at a constant but slower rate over the next few hours. The cytotoxic activity of ricin-treated cells evaluated against antigen-related target cells, in a short term incubation 51Cr release assay, was unaffected during the first 30 min after treatment but decreased with time over the next few hours. However, the growth of antigen related as well as of unrelated tumour cells was strongly inhibited by the addition of ricin-treated cells to the culture system, thus indicating that released ricin is toxic for untreated target cells. The in vivo localization pattern of ricin-treated radiolabelled MLTC cells was found to be comparable with that of untreated cells 1 h after i.v. injection into syngeneic sublethally irradiated mice. After 6 h, however, more radiolabel was recovered from the liver of mice receiving ricin-treated MLTC cells. Ricin-treated M-MSV-specific T-lymphocytes were injected i.v. into tumour bearing sublethally irradiated mice. A temporary tumour growth inhibition (up to 6 days) was achieved following transfer of low doses of ricin-treated MLTC or CTL clone cells (1 X 10(6) and 0.5 X 10(6), respectively). In contrast, in M-MSV injected control mice, receiving only free toxin (from 5 to 20 ng) or untreated tumour-specific effector cell tumours grew progressively. The therapeutic effect was apparently specific since the injection of ricin-treated alloreactive T-lymphocytes did not influence tumour growth. These results suggest that M-MSV-specific T-lymphocytes loaded with ricin can deliver toxin to the target tumour mass and have a transient therapeutic effect.

Cerundolo V, Lahaye T, Horvath C, Zanovello P, Collavo D, Engers HD. 1987. Functional activity in vivo of effector T cell populations. III. Protection against Moloney murine sarcoma virus (M-MSV)-induced tumors in T cell deficient mice by the adoptive transfer of a M-MSV-specific cytolytic T lymphocyte clone. Eur J Immunol, 17 (2), pp. 173-178. | Show Abstract | Read more

The functional activity of Moloney murine sarcoma virus (M-MSV)-specific T lymphocytes in vivo was assayed by the i.v. injection of virus-specific T lymphocytes into T cell-deficient "B mice". Virus-specific T lymphocytes generated in mixed lymphocyte tumor cell cultures were transferred i.v. into syngeneic "B mice" injected simultaneously at a distant site with the virus. These experiments indicated that a low dose (1 X 10(6) cultured cells) of infused lymphocytes can afford protection. To define the T lymphocyte subpopulation which was active, Lyt-2+ lymphocytes were selected by "panning" on plastic petri dishes coated with anti-Lyt-2 monoclonal antibody, and Lyt-2- lymphocytes selected by treatment with anti-Lyt-2 monoclonal antibody and complement. The results indicated that a Lyt-2+ lymphocyte-enriched population was more efficient in conferring protection against M-MSV-induced tumors. To investigate if cytolytic T lymphocytes (CTL) alone had a protective effect, a M-MSV-specific CTL clone was transferred in the same model system. The results demonstrated that a M-MSV-specific CTL clone prevented M-MSV-induced tumor growth and also induced the destruction of syngeneic Moloney murine leukemia virus (M-MuLV)-induced MBL-2 leukemic cells in the peritoneal cavity. However, the cell dose required to obtain protection using a CTL clone was higher than that which was effective when mixed lymphocyte tumor cell culture cells were used. To assess the ability of the transferred cells to home and to repopulate the lymphoid organs of the "B mice", the frequency of virus-specific CTL precursors in the spleen was evaluated by limiting dilution analysis. The results indicated that lymphocytes from mixed lymphocyte tumor cell cultures can be recovered from the spleens of "B mice" injected i.v. 25 days earlier. On the contrary, following the transfer of an active CTL clone, a very low frequency (less than 1/200,000 cells) of virus-specific CTL precursors was present in the spleens of recipient animals. The same M-MSV-specific CTL clone did not yield protection against M-MSV-induced tumors or MBL-2 leukemic cells when injected i.v. into M-MuLV tolerant mice.

Ronchese F, Collavo D, Zanovello P, Cerundolo V, Biasi G. 1985. Reversibility of lymphokine-induced NK-like activity in virus-specific cytotoxic T-lymphocyte clones. Immunology, 54 (2), pp. 265-274. | Show Abstract

A limiting dilution microculture system, supplemented with a source of interleukin-2 (IL-2), was employed to evaluate the frequency of Moloney-murine leukaemia/sarcoma virus (M-MuLV/M-MSV)-specific cytotoxic T-lymphocyte precursors (CTL-p) which also exhibited NK-like activity. Spleen cells, obtained from M-MuLV/M-MSV regressor mice, were restimulated in bulk secondary mixed leucocyte-tumour cell cultures (MLTC), and subsequently plated in a culture medium supplemented with two different supernatants (SN) produced following PMA-stimulation of the same EL-4 thymoma cell line. SN 20, obtained from the cell line maintained in vitro, contained IL-2 and only negligible amounts (less than 3 U/ml) of interferon (IFN), while SN 19, obtained after passage of the ascitic form of EL-4 thymoma in syngeneic mice, contained both IL-2 and IFN in high titres. The frequency of CTL-p specific for MBL-2 lymphoma cells was high and comparable in cultures supplemented with both SN (1/2 X 84 cells and 1/2 X 40 cells, respectively), while the frequency of CTL-p directed against NK-susceptible YAC-1 target cells was low in SN 20 (1/90 cells) and high in SN 19 (1/5 X 40 cells). An analysis of individual microcultures established at low cell dose (1 cell/well) indicated that specific and NK-like activity could be ascribed to the same precursor cells. Furthermore, using different long-term CTL clones, we observed that, after passage in SN 20, double-reactive clones gradually lose the capacity to lyse NK-susceptible targets, while most of MBL-2 specific clones acquired NK-like activity following a few passages in SN 19. Therefore, the induction of NK-like activity is reversible and may be modulated by soluble factors present in supernatant in which CTL clones are maintained. Double-reactive clones were unable to lyse NK-resistant allogeneic tumour cells or normal syngeneic blast cells. A few clones cross-reacting with H-2d alloantigens also exhibited NK-like activity when maintained in SN 19. The different pattern of CTL clone activity was associated with a morphological change in the clones themselves: the acquisition of double activity was accompanied by an increase in cell size and the appearance of numerous cytoplasmic granules. All CTL clones were phenotypically Thy-1+ and Lyt-2+ on indirect immunofluorescence and complement-dependent cytotoxicity investigation.(ABSTRACT TRUNCATED AT 400 WORDS)

Collavo D, Ronchese F, Zanovello P, Cerundolo V, Biasi G. 1984. Reduction in precursors of cytotoxic T lymphocytes and of cells with natural killer-like activity in spleens of cyclophosphamide-treated mice. Int J Immunopharmacol, 6 (5), pp. 529-534. | Show Abstract | Read more

The effect of cyclophosphamide (CY) on precursors of cytotoxic T lymphocytes (CTL), specific for Moloney-murine leukemia virus (M-MuL V)- induced antigens, and on the precursors of cells having natural killer (NK)-like activity was studied by means of limiting dilution assay. Pre-treatment with a single CY dose of 100 mg/kg induced a marked reduction not only in the total spleen cell number but also in the frequencies of M-MuL V-specific CTL, and NK-like cell precursors. Maximal effect was obtained 2 days after CY injection, and a gradual recovery in both total spleen cell number and cytotoxic activity was achieved by day 12. These results confirm that CY exerts a strong but transient immunodepressive effect.

Timosenko E, Ghadbane H, Silk JD, Shepherd D, Gileadi U, Howson LJ, Laynes R, Zhao Q, Strausberg RL, Olsen LR et al. 2016. Nutritional Stress Induced by Tryptophan-Degrading Enzymes Results in ATF4-Dependent Reprogramming of the Amino Acid Transporter Profile in Tumor Cells. Cancer Res, 76 (21), pp. 6193-6204. | Show Abstract | Read more

Tryptophan degradation is an immune escape strategy shared by many tumors. However, cancer cells' compensatory mechanisms remain unclear. We demonstrate here that a shortage of tryptophan caused by expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) resulted in ATF4-dependent upregulation of several amino acid transporters, including SLC1A5 and its truncated isoforms, which in turn enhanced tryptophan and glutamine uptake. Importantly, SLC1A5 failed to be upregulated in resting human T cells kept under low tryptophan conditions but was enhanced upon cognate antigen T-cell receptor engagement. Our results highlight key differences in the ability of tumor and T cells to adapt to tryptophan starvation and provide important insights into the poor prognosis of tumors coexpressing IDO and SLC1A5. Cancer Res; 76(21); 6193-204. ©2016 AACR.

Torreno-Pina JA, Manzo C, Salio M, Aichinger MC, Oddone A, Lakadamyali M, Shepherd D, Besra GS, Cerundolo V, Garcia-Parajo MF. 2016. The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells. Proc Natl Acad Sci U S A, 113 (6), pp. E772-E781. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such "tonic" activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.

Subramaniam S, Aslam A, Misbah SA, Salio M, Cerundolo V, Moody DB, Ogg G. 2016. Elevated and cross-responsive CD1a-reactive T cells in bee and wasp venom allergic individuals. Eur J Immunol, 46 (1), pp. 242-252. | Show Abstract | Read more

The role of CD1a-reactive T cells in human allergic disease is unknown. We have previously shown that circulating CD1a-reactive T cells recognize neolipid antigens generated by bee and wasp venom phospholipase, and here tested the hypothesis that venom-responsive CD1a-reactive T cells associate with venom allergy. Circulating T cells from bee and wasp venom allergic individuals, before and during immunotherapy, were exposed to CD1a-transfected K562 cells in the presence of wasp or bee venom. T-cell response was evaluated based on IFNγ, GM-CSF, and IL-13 cytokine production. Venom allergic individuals showed significantly higher frequencies of IFN-γ, GM-CSF, and IL-13 producing CD1a-reactive T cells responsive to venom and venom-derived phospholipase than healthy individuals. Venom-responsive CD1a-reactive T cells were cross-responsive between wasp and bee suggesting shared pathways of allergenicity. Frequencies of CD1a-reactive T cells were initially induced during subcutaneous immunotherapy, peaking by weeks 5, but then reduced despite escalation of antigen dose. Our current understanding of venom allergy and immunotherapy is largely based on peptide and protein-specific T cell and antibody responses. Here, we show that lipid antigens and CD1a-reactive T cells associate with the allergic response. These data have implications for mechanisms of allergy and approaches to immunotherapy.

Howson LJ, Salio M, Cerundolo V. 2015. MR1-Restricted Mucosal-Associated Invariant T Cells and Their Activation during Infectious Diseases. Front Immunol, 6 (JUN), pp. 303. | Show Abstract | Read more

MR1-restricted mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites, which are generated by a broad range of bacteria, from Escherichia coli to Mycobacterium tuberculosis and BCG. MAIT cells have been described as innate sensors of infection as they accumulate early in infected tissues. MAIT cells maintain an activated phenotype throughout the course of infections, secrete inflammatory cytokines, and have the potential to directly kill infected cells, playing an important role in shaping the host response. In this review, we will discuss the current knowledge regarding the molecular mechanisms that underline MAIT cells activation in sterile and non-sterile inflammatory conditions.

Timosenko E, Hadjinicolaou AV, Cerundolo V. 2017. Modulation of cancer-specific immune responses by amino acid degrading enzymes. Immunotherapy, 9 (1), pp. 83-97. | Show Abstract | Read more

To evade immune destruction, tumors exploit a wide range of immune escape mechanisms, including the induction of an immunosuppressive tumor microenvironment. This is mediated, in part, by amino acid degrading enzymes indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, arginase 1 and arginase 2, which have emerged as key players in the regulation of tumor-induced immune tolerance. Here we describe how the expression of tryptophan- and arginine-degrading enzymes by tumor and tumor-infiltrating cells can hamper cancer-specific immune responses, and discuss how this knowledge is being exploited to develop new strategies for cancer immunotherapy.

Cheung KL, Jarrett R, Subramaniam S, Salimi M, Gutowska-Owsiak D, Chen YL, Hardman C, Xue L, Cerundolo V, Ogg G. 2016. Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a. J Exp Med, 213 (11), pp. 2399-2412. | Show Abstract | Read more

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Jukes JP, Gileadi U, Ghadbane H, Yu TF, Shepherd D, Cox LR, Besra GS, Cerundolo V. 2016. Non-glycosidic compounds can stimulate both human and mouse iNKT cells. Eur J Immunol, 46 (5), pp. 1224-1234. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non-glycosidic CD1d-binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6- or 7-membered ring results in significantly more potent non-glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti-tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α-galactosylceramide (α-GalCer), achieving an enhanced T-cell response at lower concentrations compared with α-GalCer both in vitro, using human iNKT-cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non-glycosidic ThrCer-based analogs that have improved potency in iNKT-cell activation compared with that of α-GalCer, and are clinically relevant iNKT-cell agonists.

Kamaladasa A, Wickramasinghe N, Adikari TN, Gomes L, Shyamali NL, Salio M, Cerundolo V, Ogg GS, Malavige GN. 2016. Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection. Clin Exp Immunol, 185 (2), pp. 228-238. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)-γ and interleukin (IL)-4 ex-vivo enzyme-linked immunospot (ELISPOT) assays following stimulation with alpha-galactosyl-ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4(+) subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus-specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl-6 (P = 0·0003) and both Bcl-6 and inducible T cell co-stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4(+) iNKT cells, with reduced expression of CD161 markers.

Jarrett R, Salio M, Lloyd-Lavery A, Subramaniam S, Bourgeois E, Archer C, Cheung KL, Hardman C, Chandler D, Salimi M et al. 2016. Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase. Sci Transl Med, 8 (325), pp. 325ra18. | Show Abstract | Read more

Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation contribute to pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and although Langerhans cells expressing the nonclassical major histocompatibility complex (MHC) family member CD1a are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. We observed that house dust mite (HDM) allergen generates neolipid antigens presented by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth in individuals with atopic dermatitis and showed rapid effector function, consistent with antigen-driven maturation. In HDM-challenged human skin, we observed phospholipase A2 (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2, and such cells infiltrated the skin after allergen challenge. Moreover, we observed that the skin barrier protein filaggrin, insufficiency of which is associated with atopic skin disease, inhibited PLA2 activity and decreased CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity suggest that nonpeptide stimulants of T cells act as haptens that modify peptides or proteins; however, our results show that HDM proteins may also generate neolipid antigens that directly activate T cells. These data define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach.

Van Rhijn I, van Berlo T, Hilmenyuk T, Cheng TY, Wolf BJ, Tatituri RV, Uldrich AP, Napolitani G, Cerundolo V, Altman JD et al. 2016. Human autoreactive T cells recognize CD1b and phospholipids. Proc Natl Acad Sci U S A, 113 (2), pp. 380-385. | Show Abstract | Read more

In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids.

Nakaya HI, Clutterbuck E, Kazmin D, Wang L, Cortese M, Bosinger SE, Patel NB, Zak DE, Aderem A, Dong T et al. 2016. Systems biology of immunity to MF59-adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood. Proc Natl Acad Sci U S A, 113 (7), pp. 1853-1858. | Show Abstract | Read more

The dynamics and molecular mechanisms underlying vaccine immunity in early childhood remain poorly understood. Here we applied systems approaches to investigate the innate and adaptive responses to trivalent inactivated influenza vaccine (TIV) and MF59-adjuvanted TIV (ATIV) in 90 14- to 24-mo-old healthy children. MF59 enhanced the magnitude and kinetics of serum antibody titers following vaccination, and induced a greater frequency of vaccine specific, multicytokine-producing CD4(+) T cells. Compared with transcriptional responses to TIV vaccination previously reported in adults, responses to TIV in infants were markedly attenuated, limited to genes regulating antiviral and antigen presentation pathways, and observed only in a subset of vaccinees. In contrast, transcriptional responses to ATIV boost were more homogenous and robust. Interestingly, a day 1 gene signature characteristic of the innate response (antiviral IFN genes, dendritic cell, and monocyte responses) correlated with hemagglutination at day 28. These findings demonstrate that MF59 enhances the magnitude, kinetics, and consistency of the innate and adaptive response to vaccination with the seasonal influenza vaccine during early childhood, and identify potential molecular correlates of antibody responses.

Galson JD, Trück J, Clutterbuck EA, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2016. B-cell repertoire dynamics after sequential hepatitis B vaccination and evidence for cross-reactive B-cell activation. Genome Med, 8 (1), pp. 68. | Show Abstract | Read more

BACKGROUND: A diverse B-cell repertoire is essential for recognition and response to infectious and vaccine antigens. High-throughput sequencing of B-cell receptor (BCR) genes can now be used to study the B-cell repertoire at great depth and may shed more light on B-cell responses than conventional immunological methods. Here, we use high-throughput BCR sequencing to provide novel insight into B-cell dynamics following a primary course of hepatitis B vaccination. METHODS: Nine vaccine-naïve participants were administered three doses of hepatitis B vaccine (months 0, 1, and 2 or 7). High-throughput Illumina sequencing of the total BCR repertoire was combined with targeted sequencing of sorted vaccine antigen-enriched B cells to analyze the longitudinal response of both the total and vaccine-specific repertoire after each vaccine. ELISpot was used to determine vaccine-specific cell numbers following each vaccine. RESULTS: Deconvoluting the vaccine-specific from total BCR repertoire showed that vaccine-specific sequence clusters comprised <0.1 % of total sequence clusters, and had certain stereotypic features. The vaccine-specific BCR sequence clusters were expanded after each of the three vaccine doses, despite no vaccine-specific B cells being detected by ELISpot after the first vaccine dose. These vaccine-specific BCR clusters detected after the first vaccine dose had distinct properties compared to those detected after subsequent doses; they were more mutated, present at low frequency even prior to vaccination, and appeared to be derived from more mature B cells. CONCLUSIONS: These results demonstrate the high-sensitivity of our vaccine-specific BCR analysis approach and suggest an alternative view of the B-cell response to novel antigens. In the response to the first vaccine dose, many vaccine-specific BCR clusters appeared to largely derive from previously activated cross-reactive B cells that have low affinity for the vaccine antigen, and subsequent doses were required to yield higher affinity B cells.

Dölen Y, Kreutz M, Gileadi U, Tel J, Vasaturo A, van Dinther EA, van Hout-Kuijer MA, Cerundolo V, Figdor CG. 2016. Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses. Oncoimmunology, 5 (1), pp. e1068493. | Show Abstract | Read more

Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here, we compared the efficacy of the invariant NKT (iNKT) cell agonist α-galactosylceramide (α-GalCer) and TLR ligands (R848 and poly I:C) as an adjuvant for the full length ovalbumin (OVA) in PLGA nanoparticles. We observed that OVA+α-GalCer nanoparticles (NP) are superior over OVA+TLR-L NP in generating and stimulating antigen-specific cytotoxic T lymphocytes without the need for CD4(+) T cell help. Not only a 4-fold higher induction of antigen-specific T cells was observed, but also a more profound IFN-γ secretion was obtained by the addition α-GalCer. Surprisingly, we observed that mixtures of OVA containing NP with α-GalCer were ineffective, demonstrating that co-encapsulation of both α-GalCer and antigen within the same nanoparticle is essential for the observed T cell responses. Moreover, a single immunization with OVA+α-GalCer NP provided substantial protection from tumor formation and even delayed the growth of already established tumors, which coincided with a prominent and enhanced antigen-specific CD8(+) T cell infiltration. The provided evidence on the advantage of antigen and α-GalCer coencapsulation should be considered in the design of future nanoparticle vaccines for therapeutic purposes.

Galson JD, Trück J, Fowler A, Clutterbuck EA, Münz M, Cerundolo V, Reinhard C, van der Most R, Pollard AJ, Lunter G, Kelly DF. 2015. Analysis of B Cell Repertoire Dynamics Following Hepatitis B Vaccination in Humans, and Enrichment of Vaccine-specific Antibody Sequences. EBioMedicine, 2 (12), pp. 2070-2079. | Show Abstract | Read more

Generating a diverse B cell immunoglobulin repertoire is essential for protection against infection. The repertoire in humans can now be comprehensively measured by high-throughput sequencing. Using hepatitis B vaccination as a model, we determined how the total immunoglobulin sequence repertoire changes following antigen exposure in humans, and compared this to sequences from vaccine-specific sorted cells. Clonal sequence expansions were seen 7 days after vaccination, which correlated with vaccine-specific plasma cell numbers. These expansions caused an increase in mutation, and a decrease in diversity and complementarity-determining region 3 sequence length in the repertoire. We also saw an increase in sequence convergence between participants 14 and 21 days after vaccination, coinciding with an increase of vaccine-specific memory cells. These features allowed development of a model for in silico enrichment of vaccine-specific sequences from the total repertoire. Identifying antigen-specific sequences from total repertoire data could aid our understanding B cell driven immunity, and be used for disease diagnostics and vaccine evaluation.

Galson JD, Clutterbuck EA, Trück J, Ramasamy MN, Münz M, Fowler A, Cerundolo V, Pollard AJ, Lunter G, Kelly DF. 2015. BCR repertoire sequencing: different patterns of B-cell activation after two Meningococcal vaccines. Immunol Cell Biol, 93 (10), pp. 885-895. | Show Abstract | Read more

Next-generation sequencing was used to investigate the B-cell receptor heavy chain transcript repertoire of different B-cell subsets (naive, marginal zone (MZ), immunoglobulin M (IgM) memory and IgG memory) at baseline, and of plasma cells (PCs) 7 days following administration of serogroup ACWY meningococcal polysaccharide and protein-polysaccharide conjugate vaccines. Baseline B-cell subsets could be distinguished from each other using a small number of repertoire properties (clonality, mutation from germline and complementarity-determining region 3 (CDR3) length) that were conserved between individuals. However, analyzing the CDR3 amino-acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 PCs demonstrated nearly 10-fold greater sequence sharing between individuals than the baseline subsets, consistent with the PCs being induced by the vaccine antigen and sharing specificity for a more limited range of epitopes. By annotating PC sequences based on IgG subclass usage and mutation, and also comparing them with the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. PCs produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the PCs were predominantly IgG2, less mutated and were equally likely to be related to MZ, IgM memory or IgG memory cells. High-throughput B-cell repertoire sequencing thus provides a unique insight into patterns of B-cell activation not possible from more conventional measures of immunogenicity.

McEwen-Smith RM, Salio M, Cerundolo V. 2015. The regulatory role of invariant NKT cells in tumor immunity. Cancer Immunol Res, 3 (5), pp. 425-435. | Show Abstract | Read more

Invariant natural killer T (iNKT) cells are a unique population of T lymphocytes, which lie at the interface between the innate and adaptive immune systems, and are important mediators of immune responses and tumor surveillance. iNKT cells recognize lipid antigens in a CD1d-dependent manner; their subsequent activation results in a rapid and specific downstream response, which enhances both innate and adaptive immunity. The capacity of iNKT cells to modify the immune microenvironment influences the ability of the host to control tumor growth, making them an important population to be harnessed in the clinic for the development of anticancer therapeutics. Indeed, the identification of strong iNKT-cell agonists, such as α-galactosylceramide (α-GalCer) and its analogues, has led to the development of synthetic lipids that have shown potential in vaccination and treatment against cancers. In this Masters of Immunology article, we discuss these latest findings and summarize the major discoveries in iNKT-cell biology, which have enabled the design of potent strategies for immune-mediated tumor destruction.

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