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CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1.
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.
High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.
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.
In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.
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.
Differences in phenotype and function between spontaneously occurring melan-A-, tyrosinase- and influenza matrix peptide-specific CTL in HLA-A*0201 melanoma patients.
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.
Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors.
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.
Role of the proteasome and non proteasomal proteases in the generation of CTL epitopes
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.
DOCK8 is critical for the survival and function of NKT cells.
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.
Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.
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.
NKT-dependent B-cell activation in Gaucher disease.
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.
NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing.
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.
NAADP Activates two-pore channels on t cell cytolytic granules to stimulate exocytosis and killing
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 [1-4]. Here we identify the Ca 2+ -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 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. © 2012 Elsevier Ltd.
Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation.
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.
Exploiting retrograde transport of Shiga-like toxin 1 for the delivery of exogenous antigens into the MHC class I presentation pathway.
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.
Amide analogues of CD1d agonists modulate iNKT-cell-mediated cytokine production.
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.
Structural and kinetic basis for heightened immunogenicity of T cell vaccines.
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.
Kinetics and mechanics of two-dimensional interactions between T cell receptors and different activating ligands.
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.