Prof Graham Ogg

Research Area: Immunology
Technology Exchange: Bioinformatics, Cell sorting, Cellular immunology, Drug discovery, Flow cytometry, Immunohistochemistry, Microscopy (Confocal), Protein interaction and Vaccine production and evaluation
Keywords: T cells, atopic disease, dermatitis, varicella zoster virus, CD1a, nuocytes, filaggrin and ILC2
Web Links:
Schematic of pathogenesis of atopic eczema

Schematic of pathogenesis of atopic eczema

Keratinocytes sample their environment and can engulf fluorescent particles

Keratinocytes sample their environment and can engulf fluorescent particles

Skin and mucosae frequently represent the first point of contact with pathogens and allergens, yet we still know relatively little of the role of the surface immune system in clearing such challenges. This is crucially important in understanding the mechanisms of skin diseases and related diseases, and for optimising approaches to cutaneous drug and vaccine delivery. The aim of the group is therefore to understand, at the molecular and cellular level, the role of human cutaneous immune responses in mechanisms of disease, treatment and vaccination.  As well as contributing to an understanding of disease pathogenesis, we aim to translate our findings to changes in clinical practice.

Name Department Institution Country
Prof Vincenzo Cerundolo Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Professor Paul Klenerman Experimental Medicine Division Oxford University, Peter Medawar Building United Kingdom
Professor Persephone Borrow NDM Research Building Oxford University, NDM Research Building United Kingdom
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 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.

Salimi M, Subramaniam S, Selvakumar T, Wang X, Zemenides S, Johnson D, Ogg G. 2016. Enhanced isolation of lymphoid cells from human skin. Clin Exp Dermatol, 41 (5), pp. 552-556. | Show Abstract | Read more

Studying skin immune cells under various pathophysiological conditions is vital for understanding the nature of cutaneous inflammatory responses. Available methods of isolating cells from the skin have relatively low yield or require in vitro culture. To increase the effective isolation of skin immune cells, we used collagenase P treatment. The number of T cells obtained ex vivo using this technique was dramatically greater than that obtained with conventional methods, without the need for long-term culture. The phenotype and function of isolated cells were comparable with those of cells isolated by EDTA treatment. Collagenase P-based methods will enhance the ability to investigate lymphoid cell function in both healthy and diseased skin.

Adikari TN, Gomes L, Wickramasinghe N, Salimi M, Wijesiriwardana N, Kamaladasa A, Shyamali NL, Ogg GS, Malavige GN. 2016. Dengue NS1 antigen contributes to disease severity by inducing interleukin (IL)-10 by monocytes. Clin Exp Immunol, 184 (1), pp. 90-100. | Show Abstract | Read more

Both dengue NS1 antigen and serum interleukin (IL)-10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL-10 has also been shown to suppress dengue-specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL-10 production. Serum IL-10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL-10 levels correlated positively with dengue NS1 antigen levels (Spearman's r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearman's r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co-stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co-culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co-cultured with NS1 produced high levels of IL-10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL-10 production by monocytes.

Malavige GN, Ogg GS. 2015. Role of skin homing T cells in acute dengue infection. Ann Transl Med, 3 (17), pp. 252. | Read more

Fernando AN, Malavige GN, Perera KL, Premawansa S, Ogg GS, De Silva AD. 2015. Polymorphisms of Transporter Associated with Antigen Presentation, Tumor Necrosis Factor-α and Interleukin-10 and their Implications for Protection and Susceptibility to Severe Forms of Dengue Fever in Patients in Sri Lanka. J Glob Infect Dis, 7 (4), pp. 157-164. | Show Abstract | Read more

CONTEXT: To date, a clear understanding of dengue disease pathogenesis remains elusive. Some infected individuals display no symptoms while others develop severe life-threatening forms of the disease. It is widely believed that host genetic factors influence dengue severity. AIMS: This study evaluates the relationship between certain polymorphisms and dengue severity in Sri Lankan patients. SETTINGS AND DESIGN: Polymorphism studies are carried out on genes for; transporter associated with antigen presentation (TAP), promoter of tumor necrosis factor-α (TNF-α), and promoter of interleukin-10 (IL-10). In other populations, TAP1 (333), TAP2 (379), TNF-α (-308), and IL-10 (-1082, -819, -592) have been associated with dengue and a number of different diseases. Data have not been collected previously for these polymorphisms for dengue patients in Sri Lanka. MATERIALS AND METHODS: The polymorphisms were typed by amplification refractory mutation system polymerase chain reaction in 107 dengue hemorrhagic fever (DHF) patients together with 62 healthy controls. STATISTICAL ANALYSIS USED: Pearson's Chi-square contingency table analysis with Yates' correction. RESULTS: Neither the TAP nor the IL-10 polymorphisms considered individually can define dengue disease outcome with regard to severity. However, the genotype combination, IL-10 (-592/-819/-1082) CCA/ATA was significantly associated with development of severe dengue in these patients, suggesting a risk factor to developing DHF. Also, identified is the genotype combination IL-10 (-592/-819/-1082) ATA/ATG which suggested a possibility for protection from DHF. The TNF-α (-308) GG genotype was also significantly associated with severe dengue, suggesting a significant risk factor. CONCLUSIONS: The results reported here are specific to the Sri Lankan population. Comparisons with previous reports imply that data may vary from population to population.

Saunders SP, Moran T, Floudas A, Wurlod F, Kaszlikowska A, Salimi M, Quinn EM, Oliphant CJ et al. 2016. Spontaneous atopic dermatitis is mediated by innate immunity, with the secondary lung inflammation of the atopic march requiring adaptive immunity. J Allergy Clin Immunol, 137 (2), pp. 482-491. | Show Abstract | Read more

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin condition that can occur in early life, predisposing to asthma development in a phenomenon known as the atopic march. Although genetic and environmental factors are known to contribute to AD and asthma, the mechanisms underlying the atopic march remain poorly understood. Filaggrin loss-of-function mutations are a major genetic predisposer for the development of AD and progression to AD-associated asthma. OBJECTIVE: We sought to experimentally address whether filaggrin mutations in mice lead to the development of spontaneous eczematous inflammation and address the aberrant immunologic milieu arising in a mouse model of filaggrin deficiency. METHODS: Filaggrin mutant mice were generated on the proallergic BALB/c background, creating a novel model for the assessment of spontaneous AD-like inflammation. Independently recruited AD case collections were analyzed to define associations between filaggrin mutations and immunologic phenotypes. RESULTS: Filaggrin-deficient mice on a BALB/c background had profound spontaneous AD-like inflammation with progression to compromised pulmonary function with age, reflecting the atopic march in patients with AD. Strikingly, skin inflammation occurs independently of adaptive immunity and is associated with cutaneous expansion of IL-5-producing type 2 innate lymphoid cells. Furthermore, subjects with filaggrin mutations have an increased frequency of type 2 innate lymphoid cells in the skin in comparison with control subjects. CONCLUSION: This study provides new insights into our understanding of the atopic march, with innate immunity initiating dermatitis and the adaptive immunity required for subsequent development of compromised lung function.

Jeewandara C, Adikari TN, Gomes L, Fernando S, Fernando RH, Perera MK, Ariyaratne D, Kamaladasa A et al. 2015. Functionality of dengue virus specific memory T cell responses in individuals who were hospitalized or who had mild or subclinical dengue infection. PLoS Negl Trop Dis, 9 (4), pp. e0003673. | Show Abstract | Read more

BACKGROUND: Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus. CONCLUSIONS/SIGNIFICANCE: The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.

Vukmanovic-Stejic M, Sandhu D, Seidel JA, Patel N, Sobande TO, Agius E, Jackson SE, Fuentes-Duculan J et al. 2015. The Characterization of Varicella Zoster Virus-Specific T Cells in Skin and Blood during Aging. J Invest Dermatol, 135 (7), pp. 1752-1762. | Show Abstract | Read more

Reactivation of the varicella zoster virus (VZV) increases during aging. Although the effects of VZV reactivation are observed in the skin (shingles), the number and functional capacity of cutaneous VZV-specific T cells have not been investigated. The numbers of circulating IFN-γ-secreting VZV-specific CD4(+) T cells are significantly decreased in old subjects. However, other measures of VZV-specific CD4(+) T cells, including proliferative capacity to VZV antigen stimulation and identification of VZV-specific CD4(+) T cells with an major histocompatibility complex class II tetramer (epitope of IE-63 protein), were similar in both age groups. The majority of T cells in the skin of both age groups expressed CD69, a characteristic of skin-resident T cells. VZV-specific CD4(+) T cells were significantly increased in the skin compared with the blood in young and old subjects, and their function was similar in both age groups. In contrast, the number of Foxp3(+) regulatory T cells and expression of the inhibitory receptor programmed cell death -1 PD-1 on CD4(+) T cells were significantly increased in the skin of older humans. Therefore, VZV-specific CD4(+) T cells in the skin of older individuals are functionally competent. However, their activity may be restricted by multiple inhibitory influences in situ.

Jeewandara C, Gomes L, Wickramasinghe N, Gutowska-Owsiak D, Waithe D, Paranavitane SA, Shyamali NL, Ogg GS, Malavige GN. 2015. Platelet activating factor contributes to vascular leak in acute dengue infection. PLoS Negl Trop Dis, 9 (2), pp. e0003459. | Show Abstract | Read more

BACKGROUND: Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection. MATERIALS AND METHODS: PAF levels were initially assessed in 25 patients with acute dengue infection to determine if they were increased in acute dengue. For investigation of the kinetics of PAF, serial PAF values were assessed in 36 patients. The effect of dengue serum on tight junction protein ZO-1 was determined by using human endothelial cell lines (HUVECs). The effect of dengue serum on and trans-endothelial resistance (TEER) was also measured on HUVECs. RESULTS: PAF levels were significantly higher in patients with acute dengue (n = 25; p = 0.001) when compared to healthy individuals (n = 12). In further investigation of the kinetics of PAF in serial blood samples of patients (n = 36), PAF levels rose just before the onset of the critical phase. PAF levels were significantly higher in patients with evidence of vascular leak throughout the course of the illness when compared to those with milder disease. Serum from patients with dengue significantly down-regulated expression of tight junction protein, ZO-1 (p = 0.004), HUVECs. This was significantly inhibited (p = 0.004) by use of a PAF receptor (PAFR) blocker. Serum from dengue patients also significantly reduced TEER and this reduction was also significantly (p = 0.02) inhibited by prior incubation with the PAFR blocker. CONCLUSION: Our results suggest the PAF is likely to be playing a significant role in inducing vascular leak in acute dengue infection which offers a potential target for therapeutic intervention.

Bourgeois EA, Subramaniam S, Cheng TY, De Jong A, Layre E, Ly D, Salimi M, Legaspi A 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.

Jeewandara C, Gomes L, Paranavitane SA, Tantirimudalige M, Panapitiya SS, Jayewardene A, Fernando S, Fernando RH, Prathapan S, Ogg GS, Malavige GN. 2015. Change in Dengue and Japanese Encephalitis Seroprevalence Rates in Sri Lanka. PLoS One, 10 (12), pp. e0144799. | Show Abstract | Read more

BACKGROUND: Sri Lanka has been affected by epidemics of dengue infections for many decades and the incidence and severity of dengue infections have been rising each year. Therefore, we investigated the age stratified seroprevalence of dengue infections in order to facilitate future dengue vaccine strategies. In addition, since the symptomatic dengue infections have increased during the past few decades, we also investigated the possible association with Japanese Encephalitis Virus (JEV) antibody seropositivity with symptomatic dengue in a community cohort in Sri Lanka. METHODS: 1689 healthy individuals who were attending a primary health care facility were recruited. Dengue and JEV antibody status was determined in all individuals and JEV vaccination status was recorded. RESULTS: 1152/1689 (68.2%) individuals were seropositive for dengue and only 133/1152 (11.5%) of them had been hospitalized to due to dengue. A significant and positive correlation was observed for dengue antibody seropositivity and age in children (Spearmans R = 0.84, p = 0.002) and in adults (Spearmans R = 0.96, p = 0.004). We observed a significant rise in the age stratified seroprevalence rates in children over a period of 12 years. For instance, in year 2003 the annual seroconversion rate was 1.5% per annum, which had risen to 3.79% per annum by 2014. We also found that both adults (p<0.001) and in children (p = 0.03) who were hospitalized due to dengue were more likely to be seropositive for JEV antibodies. However, 244 (91.4%) of adults who were seropositive for JEV had not had the JEV vaccine. CONCLUSIONS: Dengue seroprevalence rates have risen significantly over the last 12 years in Sri Lanka, possibly due to increased transmission. As individuals who were hospitalized due to dengue were more likely to be seropositive for JEV, the possibility of cross-reactive assays and/or of JEV infection on immunity to the DENV and clinical disease severity should be further investigated.

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG et al. 2015. Prostaglandin D<inf>2</inf> and leukotriene E<inf>4</inf> synergize to stimulate diverse T<inf>H</inf>2 functions and T<inf>H</inf>2 cell/neutrophil crosstalk Journal of Allergy and Clinical Immunology, 135 (5), pp. 1358-1366e11. | Show Abstract | Read more

© 2015 American Academy of Allergy, Asthma & Immunology.Background Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. Objective We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. Methods The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. Results PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. Conclusions PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Adelmann K, Hegazy AN, Ogg G, Powrie F. 2014. The role of FoxP3(+) regulatory T-cells in psoriasiform skin disease IMMUNOLOGY, 143 pp. 175-175.

Paranavitane SA, Gomes L, Kamaladasa A, Adikari TN, Wickramasinghe N, Jeewandara C, Shyamali NL, Ogg GS, Malavige GN. 2014. Dengue NS1 antigen as a marker of severe clinical disease. BMC Infect Dis, 14 (1), pp. 570. | Show Abstract | Read more

BACKGROUND: Early detection of complications significantly reduces dengue associated mortality and morbidity. We set out to determine if the NS1 rapid antigen detection test could be used as a point of care test to predict severe disease. METHODS: 186 adult patients with confirmed dengue were enrolled during day 3-8 of illness. Clinical and laboratory parameters were recorded during the course of the illness and NS1 antigen levels were determined using both the Panbio dengue early ELISA (Panbio, Australia) and a NS1 rapid antigen detection kit (SD Bioline, South Korea). RESULTS: 59.1% of patients presented to hospital on day 5-6 of illness when NS1 antigen positivity was significantly (p = 0.008) associated with severe dengue (odds ratio 3.0, 95% CI 1.39 to 6.47) and the NS1 antigen levels were significantly higher (p = 0.03) in those who went on to develop shock. Serum NS1 antigen levels significantly (p < 0.0001) and inversely correlated with the total white cell counts and lymphocyte counts. The bedside NS1 test showed comparable sensitivity (97.4%) and specificity (93.7%) to the laboratory NS1 test in our setting and cohort. CONCLUSION: NS1 antigen positivity is associated with a higher risk of developing severe dengue especially when positive beyond day 5 of illness in our cohort, and while further validation studies are required, the test can therefore potentially be used as a bedside point of care test as a warning sign of severe dengue.

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG et al. 2015. Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk. J Allergy Clin Immunol, 135 (5), pp. 1358-66.e1-11. | Show Abstract | Read more

BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Gutowska-Owsiak D, Greenwald L, Watson C, Selvakumar TA, Wang X, Ogg GS. 2014. The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin British Journal of Dermatology, 171 (4), pp. 771-778. | Show Abstract | Read more

© 2014 British Association of Dermatologists.Background Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. Objectives To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. Methods Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. Results We detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-α, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. Conclusions Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention. What's already known about this topic? Keratinocytes express the histamine-synthesizing enzyme, histidine decarboxylase (HDC), under ultraviolet B and surfactant exposure. What does this study add? HDC expression in keratinocytes is increased in atopic dermatitis. Cytokines, lipopolysaccharide and house dust mites increase HDC levels; this leads to increased histamine content in keratinocytes. Endogenously expressed histamine affects keratinocyte differentiation.

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Oliphant CJ, Hwang YY, Walker JA, Salimi M, Wong SH, Brewer JM, Englezakis A, Barlow JL et al. 2014. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4<sup>+</sup> T cells potentiates type 2 immunity and promotes parasitic helminth expulsion Immunity, 41 (2), pp. 283-295. | Show Abstract | Read more

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s invivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific Tcells to instigate a dialog in which IL-2 production from Tcells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive Tcell-mediated immunity, the ILC2 and Tcell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced Tcell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity. © 2014 The Authors.

Oliphant CJ, Hwang YY, Walker JA, Salimi M, Wong SH, Brewer JM, Englezakis A, Barlow JL et al. 2014. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion. Immunity, 41 (2), pp. 283-295. | Show Abstract | Read more

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.

Gutowska-Owsiak D, Greenwald L, Watson C, Selvakumar TA, Wang X, Ogg GS. 2014. The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin. Br J Dermatol, 171 (4), pp. 771-778. | Show Abstract | Read more

BACKGROUND: Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. OBJECTIVES: To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. METHODS: Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. RESULTS: We detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-α, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. CONCLUSIONS: Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention.

Marwah I, Wang X, Chan H, Ogg GS, Gutowska-Owsiak D. 2014. Filaggrin-insufficiency in keratinocytes influences responsiveness of allergen-specific T cells to cognate antigen and compounds barrier function deficiency. Clin Immunol, 153 (1), pp. 153-155. | Read more

Gutowska-Owsiak D, Selvakumar TA, Salimi M, Taylor S, Ogg GS. 2014. Histamine enhances keratinocyte-mediated resolution of inflammation by promoting wound healing and response to infection BRITISH JOURNAL OF DERMATOLOGY, 170 (4), pp. E31-E32.

Salimi M, Barlow J, Saunders S, Xue L, Gutowska-Owsiak D, Wang X, Huang L-C, Johnson D et al. 2014. The role of type 2 innate lymphoid cells in the pathogenesis of atopic dermatitis BRITISH JOURNAL OF DERMATOLOGY, 170 (4), pp. E32-E32.

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Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie ANJ, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells Journal of Allergy and Clinical Immunology, 133 (4), | Show Abstract | Read more

Background Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on T H2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. Objectives We sought to determine the role of PGD2 and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. Methods The effects of PGD 2, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD2 under physiologic conditions were evaluated by using the supernatant from activated mast cells. Results PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD 2 on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Conclusions PGD2 is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s. © 2013 American Academy of Allergy, Asthma & Immunology.

Gutowska-Owsiak D, Selvakumar TA, Salimi M, Taylor S, Ogg GS. 2014. Histamine enhances keratinocyte-mediated resolution of inflammation by promoting wound healing and response to infection Clinical and Experimental Dermatology, 39 (2), pp. 187-195. | Show Abstract | Read more

Background The role of the epidermis in the immune response is well known. While multiple cytokines are implicated in keratinocyte-mediated infection clearance and wound healing, little is known about the involvement of keratinocytes in promoting resolution of inflammation. Aim To assess effects of histamine stimulation on keratinocyte function. Methods We performed a combined microarray/Gene Ontology analysis of histamine-stimulated keratinocytes. Functional changes were tested by apoptosis assessment and scratch assays. Histamine receptor involvement was also assessed by blocking wound closure with specific antagonists. Results Histamine treatment had extensive effects on keratinocytes, including effects on proinflammatory responses and cellular functions promoting wound healing. At the functional level, there was reduced apoptosis and enhancement of wound healing in vitro. At the receptor level, we identified involvement of all keratinocyte-expressed histamine receptors (HRHs), with HRH1 blockage resulting in the most prominent effect. Conclusions Histamine activates wound healing and infection clearance-related functions of keratinocytes. While enhancement of histamine-mediated wound healing is mediated predominantly via the HRH1 receptor, other keratinocyte-expressed receptors are also involved. These effects could promote resolution of skin inflammation caused by infection or superficial injury. © 2014 British Association of Dermatologists.

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.

Gutowska-Owsiak D, Selvakumar TA, Salimi M, Taylor S, Ogg GS. 2014. Histamine enhances keratinocyte-mediated resolution of inflammation by promoting wound healing and response to infection. Clin Exp Dermatol, 39 (2), pp. 187-195. | Show Abstract | Read more

BACKGROUND: The role of the epidermis in the immune response is well known. While multiple cytokines are implicated in keratinocyte-mediated infection clearance and wound healing, little is known about the involvement of keratinocytes in promoting resolution of inflammation. AIM: To assess effects of histamine stimulation on keratinocyte function. METHODS: We performed a combined microarray/Gene Ontology analysis of histamine-stimulated keratinocytes. Functional changes were tested by apoptosis assessment and scratch assays. Histamine receptor involvement was also assessed by blocking wound closure with specific antagonists. RESULTS: Histamine treatment had extensive effects on keratinocytes, including effects on proinflammatory responses and cellular functions promoting wound healing. At the functional level, there was reduced apoptosis and enhancement of wound healing in vitro. At the receptor level, we identified involvement of all keratinocyte-expressed histamine receptors (HRHs), with HRH1 blockage resulting in the most prominent effect. CONCLUSIONS: Histamine activates wound healing and infection clearance-related functions of keratinocytes. While enhancement of histamine-mediated wound healing is mediated predominantly via the HRH1 receptor, other keratinocyte-expressed receptors are also involved. These effects could promote resolution of skin inflammation caused by infection or superficial injury.

Salimi M, Ogg G. 2014. Innate lymphoid cells and the skin. BMC Dermatol, 14 (1), pp. 18. | Show Abstract | Read more

Innate lymphoid cells are an emerging family of effector cells that contribute to lymphoid organogenesis, metabolism, tissue remodelling and protection against infections. They maintain homeostatic immunity at barrier surfaces such as lung, skin and gut (Nature 464:1367-1371, 2010, Nat Rev Immunol 13: 145-149, 2013). Several human and mouse studies suggest a role for innate lymphoid cells in inflammatory skin conditions including atopic eczema and psoriasis. Here we review the innate lymphoid cell family and discuss their function in the skin and during inflammation.

Gomes L, Fernando S, Fernando RH, Wickramasinghe N, Shyamali NL, Ogg GS, Malavige GN. 2014. Sphingosine 1-phosphate in acute dengue infection. PLoS One, 9 (11), pp. e113394. | Show Abstract | Read more

BACKGROUND: Vascular leak is the hallmark of severe dengue infections and leads to complications such as shock and multi-organ failure. Although many mediators have been implicated in the vascular leak in dengue, the role of sphingosine 1-phosphate (S1P) has not been investigated. METHOLODOLOGY/PRINCIPAL FINDINGS: As S1P has been shown to be important in barrier integrity, we assessed the S1P levels in 28 patients with acute dengue and 12 healthy individuals. The S1P levels were significantly lower in patients with acute dengue (p = 0.002) and the levels in patients with grade IV dengue haemorrhagic fever (DHF) were significantly lower than those with dengue fever (p = 0.005). We then investigated the kinetics of S1P levels throughout the course of the illness in another 32 patients in serum samples obtained twice a day. We found that S1P levels were low throughout the course of illness and S1P levels were <0.5 µM in 12/23 patients with DHF when compared to 1/9 with DF. CONCLUSIONS/SIGNIFICANCE: As S1P has shown to be important in the endothelial barrier integrity and increases transendothelial resistance, low levels of S1P in acute dengue infection are likely to contribute to increased vascular permeability.

Gutowska-Owsiak D, Salimi M, Selvakumar TA, Wang X, Taylor S, Ogg GS. 2014. Histamine exerts multiple effects on expression of genes associated with epidermal barrier function. J Investig Allergol Clin Immunol, 24 (4), pp. 231-239. | Show Abstract

BACKGROUND: The role of epidermal barrier genes in the pathogenesis of atopic skin inflammation has recently been highlighted. Cytokines that are abundant in the skin during inflammation have been shown to exert various effects on the expression of barrier genes, although the role of histamine in this area of skin biology is not yet fully understood. OBJECTIVE: To assess the effect of stimulation with histamine on keratinocytes by analysis of the pathways involved in epidermal barrier integrity. MATERIAL AND METHODS: We performed a gene expression analysis of histamine-stimulated keratinocytes. Functional changes were tested using the dye penetration assay. Differential changes in filaggrin and the filaggrin-processing enzyme bleomycin hydrolase (BLMH) were validated at the protein level, and expression was also assessed in filaggrin knock-down keratinocytes. RESULTS: Histamine altered expression of multiple barrier genes. Expression of filaggrin was downregulated, as was that of other markers, thus suggesting the presence of delayed/aberrant keratinocyte differentiation. Expression of genes involved in cellular adhesiveness and genes of protease expression was dysregulated, but expression of protease inhibitors was increased. BLMH was upregulated in keratinocytes subjected to histamine and filaggrin knockdown. CONCLUSIONS: Histamine exerts a dual effect on epidermal barrier genes; it suppresses keratinocyte differentiation and dysregulates genes of cellular adhesiveness, although it induces genes contributing to stratum corneum function. Upregulation of BLMH and protease inhibitors could support maintenance of the permeability barrier by enhanced generation of moisturizing compounds and suppressed desquamation. In contrast, in the case of stratum corneum damage, histamine could enhance transcutaneous sensitization.

Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie AN, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells. J Allergy Clin Immunol, 133 (4), pp. 1184-1194. | Show Abstract | Read more

BACKGROUND: Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. OBJECTIVES: We sought to determine the role of PGD₂ and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. METHODS: The effects of PGD₂, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD₂ under physiologic conditions were evaluated by using the supernatant from activated mast cells. RESULTS: PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD₂ on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. CONCLUSIONS: PGD₂ is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.

Salimi M, Barlow JL, Saunders SP, Xue L, Gutowska-Owsiak D, Wang X, Huang LC, Johnson D et al. 2013. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J Exp Med, 210 (13), pp. 2939-2950. | Show Abstract | Read more

Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.

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.

Malavige GN, Ogg GS. 2013. T cell responses in dengue viral infections Journal of Clinical Virology, 58 (4), pp. 605-611. | Show Abstract | Read more

Dengue viral infections are the commonest mosquito borne viral infection in the world, affecting more than 100 countries and 390 million individuals annually. Currently, there are no effective antiviral drugs or an effective vaccine to prevent infection. A main hurdle in developing a safe and effective vaccine has been our poor understanding of the complex nature of the protective immune response in acute dengue infection and the presence of four dengue virus (DV) serotypes that are highly homologous. The role of DV specific T cells in the pathogenesis of severe clinical disease in not clear. It has been speculated that highly cross reactive T cells for the previous infecting heterologous DV serotype, which produce pro-inflammatory cytokines, contribute to disease pathogenesis. These cross reactive T cells are believed to be suboptimal in clearing the infection with the current DV-serotype. However, other studies have shown that cross-reactive DV-specific T cells are absent or present in very low frequency during acute infection, appearing only during the convalescent period in the majority of patients. Furthermore, significant apoptosis of T cells occurs in severe acute clinical disease. Overall therefore, it is unclear what role T cells play in contributing to disease pathogenesis during acute dengue infection. Existing data have been complicated by cross-reactivity in T cells assays. These findings can now be re-evaluated in the light of novel technologies to identify serotype-specific T cell responses. © 2013 Elsevier B.V.

Malavige GN, Ogg GS. 2013. T cell responses in dengue viral infections. J Clin Virol, 58 (4), pp. 605-611. | Show Abstract | Read more

Dengue viral infections are the commonest mosquito borne viral infection in the world, affecting more than 100 countries and 390 million individuals annually. Currently, there are no effective antiviral drugs or an effective vaccine to prevent infection. A main hurdle in developing a safe and effective vaccine has been our poor understanding of the complex nature of the protective immune response in acute dengue infection and the presence of four dengue virus (DV) serotypes that are highly homologous. The role of DV specific T cells in the pathogenesis of severe clinical disease in not clear. It has been speculated that highly cross reactive T cells for the previous infecting heterologous DV serotype, which produce pro-inflammatory cytokines, contribute to disease pathogenesis. These cross reactive T cells are believed to be suboptimal in clearing the infection with the current DV-serotype. However, other studies have shown that cross-reactive DV-specific T cells are absent or present in very low frequency during acute infection, appearing only during the convalescent period in the majority of patients. Furthermore, significant apoptosis of T cells occurs in severe acute clinical disease. Overall therefore, it is unclear what role T cells play in contributing to disease pathogenesis during acute dengue infection. Existing data have been complicated by cross-reactivity in T cells assays. These findings can now be re-evaluated in the light of novel technologies to identify serotype-specific T cell responses.

Malavige GN, Gomes L, Alles L, Chang T, Salimi M, Fernando S, Nanayakkara KD, Jayaratne S, Ogg GS. 2013. Serum IL-10 as a marker of severe dengue infection. BMC Infect Dis, 13 (1), pp. 341. | Show Abstract | Read more

BACKGROUND: Several studies have shown that serum IL-10, IFNγ and MIF are elevated in patients in severe dengue (SD) and could be used as potential biomarkers. We proceeded to determine if these cytokines could be used as biomarkers in a large cohort of adult dengue patients with varying severity of dengue infection. METHODS: Serum IL-10 levels were determined in 259 of whom 40 had severe dengue infection. Serum IFNγ and IFNα levels were done in 78 and MIF levels were done in 65 patients with acute dengue infection. Clinical features and laboratory investigations were undertaken during the febrile and critical phase. RESULTS: We found that serum IL-10 levels were significantly higher (p = 0.001) in patients with SD, when compared to those with non SD. Serum IL-10 levels significantly and inversely correlated with white cell counts (R = -0.23, p = 0.0002) and lymphocyte counts (R = -0.29, p < 0.0001) but significantly and positively correlated with aspartate tranaminase levels (R = 0.16, p = 0.01) and alanine transaminase levels (R = 0.22, p = 0.007). However, IL-10 levels did not have a good predictive value in discriminating those who were likely to develop SD (AUC = 0.66). Serum IFNγ levels were also significantly higher (p = 0.04) in patients with SD when compared to non SD. There was no difference (p = 0.34) in serum IFNα levels and serum MIF levels (p = 0.15) in patients with SD and non SD. CONCLUSION: Although serum IL-10 was significantly elevated in patients with SD it had a poor discriminatory value in identifying those with SD and non SD and therefore, is unsuitable to be used as a robust biomarker in this cohort.

Gutowska-Owsiak D, Ogg GS. 2013. Cytokine regulation of the epidermal barrier. Clin Exp Allergy, 43 (6), pp. 586-598. | Show Abstract | Read more

Studies published in recent years have highlighted the role of epidermal barrier defects in both atopic skin disease and the development of broader allergic manifestations. While genetic determinants of barrier function are important, it is clear that local acquired effects are also involved in disease pathogenesis. In this review, we aimed to summarize the known influences of cytokines abundantly expressed during atopic skin disease on components of epidermal barrier integrity and function.

Huang LC, Pan X, Yang H, Wan LK, Stewart-Jones G, Dorrell L, Ogg G. 2013. Linking genotype to phenotype on beads: high throughput selection of peptides with biological function. Sci Rep, 3 pp. 3030. | Show Abstract | Read more

Although peptides are well recognised biological molecules in vivo, their selection from libraries is challenging because of relative low affinity whilst in linear conformation. We hypothesized that multiplexed peptides and DNA on the surface of beads would provide a platform for enhanced avidity and the selection of relevant peptides from a library (ORBIT bead display). Using human immunodeficiency virus (HIV-1) gp120 as a target, we identify peptides that inhibit HIV-1 replication in vitro through blocking of protein:protein interaction with the co-receptor CCR5. The bead display approach has many potential applications for probing biological systems and for drug lead development.

Malavige GN, Jeewandara C, Alles KM, Salimi M, Gomes L, Kamaladasa A, Jayaratne SD, Ogg GS. 2013. Suppression of virus specific immune responses by IL-10 in acute dengue infection. PLoS Negl Trop Dis, 7 (9), pp. e2409. | Show Abstract | Read more

BACKGROUND: Elevated IL-10 has been shown to be associated with severe dengue infection (DI). We proceeded to investigate the role of IL-10 in the pathogenesis of acute DI. MATERIALS AND METHODS: Ex vivo and cultured IFNγ ELISpot assays for dengue virus (DENV) NS3 protein and non dengue viral proteins were carried out in 26 patients with acute DI (16 with dengue haemorrhagic fever) and 12 healthy dengue seropositive individuals from Sri Lanka. DENV serotype specific (SS) responses were determined by using a panel of SS peptides. RESULTS: Serum IL-10 level were significantly higher (p = 0.02) in those who did not have in vitro responses to DENV-SS peptides (mean 144.2 pg/ml) when compared to those who responded (mean 75.7 pg/ml). DENV-NS3 specific ex vivo IFNγ ELISpot responses were also significantly lower (p = 0.0001) in those who did not respond to DENV-SS peptides (mean 42 SFU/million PBMCs) when compared to those who responded to DENV-SS peptides (mean 1024 SFU/million PBMCs). Serum IL-10 levels correlated significantly (p = 0.03) and inversely (Spearmans R = -0.45) with ex vivo DENV-NS3 specific responses but not with ex vivo non DENV specific responses (Spearmans R = -014, p = 0.52). Blockage of IL-10 in vitro significantly increased (p = 0.04) the ex vivo IFNγ ELISpot DENV-NS3 specific responses but had no effect on responses to non DENV proteins. CONCLUSION: IL-10 appears to contribute to the pathogenesis of acute dengue infections by inhibiting DENV-specific T cell responses, which can be restored by blocking IL-10.

Vukmanovic-Stejic M, Sandhu D, Sobande TO, Agius E, Lacy KE, Riddell N, Montez S, Dintwe OB et al. 2013. Varicella zoster-specific CD4+Foxp3+ T cells accumulate after cutaneous antigen challenge in humans. J Immunol, 190 (3), pp. 977-986. | Show Abstract | Read more

We investigated the relationship between varicella zoster virus (VZV)-specific memory CD4(+) T cells and CD4(+)Foxp3(+) regulatory T cells (Tregs) that accumulate after intradermal challenge with a VZV skin test Ag. VZV-specific CD4(+) T cells were identified with a MHC class II tetramer or by intracellular staining for either IFN-γ or IL-2 after Ag rechallenge in vitro. VZV-specific T cells, mainly of a central memory (CD45RA(-)CD27(+)) phenotype, accumulate at the site of skin challenge compared with the blood of the same individuals. This resulted in part from local proliferation because >50% of tetramer defined Ag-specific CD4(+) T cells in the skin expressed the cell cycle marker Ki67. CD4(+)Foxp3(+) T cells had the characteristic phenotype of Tregs, namely CD25(hi)CD127(lo)CD39(hi) in both unchallenged and VZV challenged skin and did not secrete IFN-γ or IL-2 after antigenic restimulation. The CD4(+)Foxp3(+) T cells from unchallenged skin had suppressive activity, because their removal led to an increase in cytokine secretion after activation. After VZV Ag injection, Foxp3(+)CD25(hi)CD127(lo)CD39(hi) T cells were also found within the VZV tetramer population. Their suppressive activity could not be directly assessed by CD25 depletion because activated T cells in the skin were also CD25(+). Nevertheless, there was an inverse correlation between decreased VZV skin responses and proportion of CD4(+)Foxp3(+) T cells present, indicating indirectly their inhibitory activity in vivo. These results suggest a linkage between the expansion of Ag-specific CD4(+) T cells and CD4(+) Tregs that may provide controlled responsiveness during Ag-specific stimulation in tissues.

Crack LR, Jones L, Malavige GN, Patel V, Ogg GS. 2012. Human antimicrobial peptides LL-37 and human β-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus. Clin Exp Dermatol, 37 (5), pp. 534-543. | Show Abstract | Read more

BACKGROUND: There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human β-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). AIM: To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human β-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. METHODS: Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. RESULTS: LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Pre-incubation of VZV with LL-37, but not hBD-2, further reduced VZV load. CONCLUSIONS: Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE.

Malavige GN, McGowan S, Atukorale V, Salimi M, Peelawatta M, Fernando N, Jayaratne SD, Ogg G. 2012. Identification of serotype-specific T cell responses to highly conserved regions of the dengue viruses. Clin Exp Immunol, 168 (2), pp. 215-223. | Show Abstract | Read more

Determining previous infecting dengue virus (DENV) serotypes has been difficult due to highly cross-reactive immune responses from previous DENV infections. Determining the correlates of serotype-specific immune responses would be crucial in understanding dengue transmission in the community and would also help to determine the correlates of protective immune responses. Therefore, we set out to define highly conserved, serotype-specific regions of the DENVs. Serotype-specific and highly conserved regions of the four DENV serotypes were identified using Basic Local Alignment Search Tool (BLAST) searches and custom perl scripts. Using ex-vivo and cultured enzyme-linked immunospot (ELISPOT) assays, we identified serotype-specific T cell epitopes within the four DENV serotypes in healthy adult donors from Sri Lanka. We identified T cell responses to 19 regions of the four DENV serotypes. Six peptides were from the NS2A region and four peptides were from the NS4A region. All immune donors responded to peptides of at least two DENV serotypes, suggesting that heterologous infection is common in Sri Lanka. Eight of 20 individuals responded to at least two peptides of DENV-4, despite this serotype not being implicated previously in any of the epidemics in Sri Lanka. The use of these regions to determine past and current infecting DENV serotypes will be of value to characterize further the dynamics of silent dengue transmission in the community. In addition, these T cell responses to these regions could be used to characterize DENV serotype-specific immune responses and thus possibly help us to understand the immune correlates of a protective immune response.

Gutowska-Owsiak D, Schaupp AL, Salimi M, Selvakumar TA, McPherson T, Taylor S, Ogg GS. 2012. IL-17 downregulates filaggrin and affects keratinocyte expression of genes associated with cellular adhesion. Exp Dermatol, 21 (2), pp. 104-110. | Show Abstract | Read more

Atopic eczema and psoriasis are common skin diseases. While it is well established that the pathogenesis of these diseases varies, both are characterized by impairment in epidermal barrier function and abnormal IL-17 expression in the skin and peripheral blood. Recent findings indicated that filaggrin is essential during barrier formation and its insufficiency underlies the pathogenesis of atopic eczema. Filaggrin downregulation has also been reported in psoriasis. It is clear that Th1/Th2 bias influences expression of the protein, but an analysis of the effects of interleukin-17 (IL-17) on the expression of the protein and profilaggrin-processing enzymes has not yet been reported. In addition, the effect of the cytokine on components of functional epidermal barrier, tight junctions and adhesion/desmosomal proteins, has not been elucidated. Keratinocytes were exposed to interleukin-17A, and microarray analysis was performed. Filaggrin protein level was assessed by western blot. We have observed a significant decrease in profilaggrin mRNA level in interleukin-17A-exposed cultures (P = 0.008). Expression of processing enzymes was also altered, indicating an indirect effect of the cytokine on filaggrin production/degradation. Moreover, expression of many genes involved in cellular adhesion was also decreased. A significant downregulation of filaggrin at the protein level was detected by western blot in immortal and primary keratinocytes. Gene ontology analysis indicated changes in keratinization, epidermal differentiation and formation of the cornified envelope. We conclude that IL-17A downregulates the expression of filaggrin and genes important for cellular adhesion which could affect epidermal barrier formation. This effect potentially contributes to barrier dysfunction and could become a possible therapeutic target.

Gutowska-Owsiak D, Ogg GS. 2012. The epidermis as an adjuvant. J Invest Dermatol, 132 (3 Pt 2), pp. 940-948. | Show Abstract | Read more

It is now clear that the epidermis has an active role in local immune responses in the skin. Keratinocytes are involved early in inflammation by providing first-line innate mechanisms and, in addition, can contribute to adaptive immune responses that may be associated with clinical disease. Moreover, keratinocytes are capable of enhancing and shaping the outcome of inflammation in response to stimuli and promoting particular types of immune bias. Through understanding the underlying mechanisms, the role of keratinocytes in disease pathogenesis will be further defined, which is likely to lead to the identification of potential targets for prophylactic or therapeutic intervention.

Malavige GN, Huang LC, Salimi M, Gomes L, Jayaratne SD, Ogg GS. 2012. Cellular and cytokine correlates of severe dengue infection. PLoS One, 7 (11), pp. e50387. | Show Abstract | Read more

BACKGROUND: The occurrence of dengue haemorrhagic fever (DHF) is thought to result from a complex interplay between the virus, host genetics and host immune factors. Existing published data are not consistent, in part related to relatively small sample sizes. We set out to determine possible associations between dengue virus (DEN-V) NS3 specific T cells and cytokine and chemokine levels and the pathogenesis of severe disease in a large cohort of individuals with DHF. METHODOLOGY/PRINCIPAL FINDINGS: By using ex vivo IFNγ ELISpot assays we determined DENV-NS3 specific responses in patients with varying severity of DHF. Other cytokines produced by DENV-NS3 specific T cells were determined by using multiple bead array analysis (MBAA). We also determined the serum cytokine levels using MBAA, lymphocyte subsets and Annexin V expression of lymphocytes in patients with varying severity of DHF. Of the 112 DHF patients studied, 29 developed shock. Serum IL-10 and IP-10 levels positively and significantly correlated with T cell apoptosis while IL-10 levels inversely correlated with T cell numbers. In contrast, TGFß showed a very significant (P<0.0001) and positive correlation (Spearman's R = 0.65) with the platelet counts, consistent with platelet release. We found that whilst patients with severe dengue had lower total T cell numbers, the DV-NS3 specific T cells persisted and produced high levels of IFNγ but not TNFα, IL-3, IL-13, IL-2, IL-10 or IL-17. CONCLUSIONS/SIGNIFICANCE: Our data suggest that serum IL-10, TNFα and TGFβ differentially associate with dengue disease severity.

Crack LR, Jones L, Malavige GN, Patel V, Ogg GS. 2012. Human antimicrobial peptides LL-37 and human β-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus Clinical and Experimental Dermatology, 37 (5), pp. 534-543. | Show Abstract | Read more

Background. There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human β-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). Aim. To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human β-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. Methods. Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. Results. LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Preincubation of VZV with LL-37, but not hBD-2, further reduced VZV load. Conclusions. Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE. © The Author(s) CED © 2012 British Association of Dermatologists.

Crack LR, Chan HW, McPherson T, Ogg GS. 2012. Identification of an immunodominant region of the major house dust mite allergen der p 2 presented by common human leucocyte antigen alleles Clinical and Experimental Dermatology, 37 (3), pp. 266-276. | Show Abstract | Read more

Background. Better understanding of the relevance of the immune response to common environmental allergens, such as the major house dust mite (HDM) allergen Der p 2, requires characterization of constituent T-cell epitopes. Aim. To identify CD4 + T-cell epitopes within Der p 2 recognized by commonly expressed human leucocyte antigen (HLA) alleles. Methods. HLA-blocking antibodies, peptide pools and truncations were used in ELISpot assays to establish restricted T-cell epitopes. Results. People with and without atopic dermatitis have detectable Der p 2-specific T cells in the peripheral blood, which can proliferate in response to Der p 2 peptides. Interleukin-4-specific responses, both ex vivo and cultured to Der p 2 peptides, had a significant positive correlation with HDM-specific serum IgE. Within one pool of Der p 2 peptides, the 20mer D11 was found to induce multiple responses restricted through several alleles, including HLA-DPB1*0401 and HLA-DRB1*01. Conclusions. We have identified an immunogenic region of Der p 2 presented by common HLA class II alleles, including the most commonly expressed HLA allele DPB1*0401. Identification of such epitopes may be of future value in peptide immunotherapeutic approaches. © 2011 British Association of Dermatologists.

Cited:

32

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Gutowska-Owsiak D, Ogg GS. 2012. The epidermis as an adjuvant Journal of Investigative Dermatology, 132 (3 PART 2), pp. 940-948. | Show Abstract | Read more

It is now clear that the epidermis has an active role in local immune responses in the skin. Keratinocytes are involved early in inflammation by providing first-line innate mechanisms and, in addition, can contribute to adaptive immune responses that may be associated with clinical disease. Moreover, keratinocytes are capable of enhancing and shaping the outcome of inflammation in response to stimuli and promoting particular types of immune bias. Through understanding the underlying mechanisms, the role of keratinocytes in disease pathogenesis will be further defined, which is likely to lead to the identification of potential targets for prophylactic or therapeutic intervention. © 2012 The Society for Investigative Dermatology.

Crack LR, Chan HW, McPherson T, Ogg GS. 2012. Identification of an immunodominant region of the major house dust mite allergen Der p 2 presented by common human leucocyte antigen alleles. Clin Exp Dermatol, 37 (3), pp. 266-276. | Show Abstract | Read more

BACKGROUND: Better understanding of the relevance of the immune response to common environmental allergens, such as the major house dust mite (HDM) allergen Der p 2, requires characterization of constituent T-cell epitopes. AIM: To identify CD4(+) T-cell epitopes within Der p 2 recognized by commonly expressed human leucocyte antigen (HLA) alleles. METHODS: HLA-blocking antibodies, peptide pools and truncations were used in ELISpot assays to establish restricted T-cell epitopes. RESULTS: People with and without atopic dermatitis have detectable Der p 2-specific T cells in the peripheral blood, which can proliferate in response to Der p 2 peptides. Interleukin-4-specific responses, both ex vivo and cultured to Der p 2 peptides, had a significant positive correlation with HDM-specific serum IgE. Within one pool of Der p 2 peptides, the 20mer D11 was found to induce multiple responses restricted through several alleles, including HLA-DPB1*0401 and HLA-DRB1*01. CONCLUSIONS: We have identified an immunogenic region of Der p 2 presented by common HLA class II alleles, including the most commonly expressed HLA allele DPB1*0401. Identification of such epitopes may be of future value in peptide immunotherapeutic approaches.

Xue L, Barrow A, Fleming VM, Hunter MG, Ogg G, Klenerman P, Pettipher R. 2012. Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine production in response to prostaglandin D2. J Immunol, 188 (2), pp. 694-702. | Show Abstract | Read more

PGD(2) exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD(2). PGD(2) induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D(4) and E(4) (LTE(4)) also stimulated the cytokine production but were much less active than PGD(2). However, when combined with PGD(2), cysLTs caused a greater than additive enhancement of the response, with LTE(4) being most effective in activating Th2 cells. LTE(4) enhanced calcium mobilization in response to PGD(2) in Th2 cells without affecting endogenous PGD(2) production or CRTH2 receptor expression. The effect of LTE(4) was inhibited by montelukast but not by the P2Y(12) antagonist methylthioadenosine 5'-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by MK886, an inhibitor of 5-lipoxygenase-activating protein, reduced the response of Th2 cells to the levels produced by PGD(2) alone. These findings reveal that cysLTs, in particular LTE(4), have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor.

Crack LR, Chan HW, McPherson T, Ogg GS. 2011. Phenotypic analysis of perennial airborne allergen-specific CD4+ T cells in atopic and non-atopic individuals. Clin Exp Allergy, 41 (11), pp. 1555-1567. | Show Abstract | Read more

BACKGROUND: Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. OBJECTIVE: To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. METHODS: Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. RESULTS: Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals.

Gutowska-Owsiak D, Schaupp AL, Salimi M, Taylor S, Ogg GS. 2011. Interleukin-22 downregulates filaggrin expression and affects expression of profilaggrin processing enzymes. Br J Dermatol, 165 (3), pp. 492-498. | Show Abstract | Read more

BACKGROUND: The identification of filaggrin mutations has contributed towards our understanding of hereditary factors associated with epidermal dysfunction observed in individuals with atopic eczema (AE). However, factors that predispose to acquired filaggrin modulation are not well understood. Interleukin (IL)-22 is upregulated in lesional AE tissue, but its effects on filaggrin expression and genes associated with epidermal function have not yet been comprehensively addressed. OBJECTIVES: To investigate the effects of IL-22 on expression of filaggrin and genes encoding proteins relevant to epidermal function. METHODS: Microarray analysis was performed on IL-22-stimulated HaCaT keratinocytes. Filaggrin protein level was assessed by an intracellular enzyme-linked immunosorbent assay (ELISA) and Western blot in HaCaT cells and the findings were validated in primary keratinocytes. RESULTS: Exposure to IL-22 cytokine resulted in a downregulation of profilaggrin mRNA expression in HaCaT keratinocytes. The expression of genes involved in enzymatic processing of profilaggrin as well as the generation of natural moisturizing factor was also altered. Furthermore, there was an upregulation of many transcripts encoding proteins of the S100 family. Profilaggrin/filaggrin downregulation was detected by intracellular ELISA and Western blot in HaCaT cells. The relevance to the primary setting was confirmed in primary keratinocytes by Western blot. CONCLUSIONS: IL-22 downregulates profilaggrin/filaggrin expression in keratinocytes at both mRNA and protein levels and affects genes relevant to epidermal function. This novel pathway may have relevance to the pathogenesis and treatment of atopic and other skin disease.

Chan HW, Aslam A, Lee CH, Jones L, Wynne K, Wright G, Crack L, McPherson T, Stauss H, Price DR, Ogg GS. 2011. Antigen dose and keratinocyte antigen presentation are specific determinants of T cell function in atopic eczema AUSTRALASIAN JOURNAL OF DERMATOLOGY, 52 pp. 19-20.

Aly L, Yousef S, Schippling S, Jelcic I, Breiden P, Matschke J, Schulz R, Bofill-Mas S et al. 2011. Central role of JC virus-specific CD4+ lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome. Brain, 134 (Pt 9), pp. 2687-2702. | Show Abstract | Read more

Progressive multi-focal leucoencephalopathy and progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are caused by infection of the central nervous system with the JC polyoma virus. Both are complications of monoclonal antibody therapy in multiple sclerosis and other autoimmune diseases. Progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome can obscure the diagnosis of progressive multi-focal leucoencephalopathy and lead to severe clinical disability and possibly death. Different from progressive multi-focal leucoencephalopathy, in which demyelination results from oligodendrocyte lysis by JC virus in the absence of an immune response, tissue destruction in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome is caused by a vigorous immune response within the brain. The cells and mediators that are involved in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are as yet poorly understood. We examined two patients with multiple sclerosis, who developed progressive multi-focal leucoencephalopathy and later progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome under natalizumab therapy. Due to initially negative JC viral deoxyribonucleic acid testing in the cerebrospinal fluid, a diagnostic brain biopsy was performed in one patient. Histopathology revealed brain inflammation characterized by a prominent T cell infiltrate (CD4(+)> CD8(+) T cells), but also B/plasma cells and monocytes. Despite very low JC viral load, both patients showed high intrathecal anti-JC virus antibodies. Brain-infiltrating CD4(+) T cells were studied regarding antigen specificity and function. CD4(+) T cells were highly specific for peptides from several JC virus proteins, particularly the major capsid protein VP1. T cell phenotyping revealed CD4(+) Th1 and bifunctional Th1-2 cells. The latter secrete large amounts of interferon-γ and interleukin-4 explaining the strong brain inflammation, presence of plasma cells and secretion of intrathecal anti-VP1 antibodies. The functional phenotype of brain-infiltrating JC virus-specific CD4(+) T cells was confirmed and extended by examining brain-derived JC virus-specific CD4(+) T cell clones. Our data provide novel insight into the pathogenesis of progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome and indicate that JC virus-specific CD4(+) T cells play an important role in both eliminating JC virus from the brain, but also in causing the massive inflammation with often fatal outcome.

Aslam A, Chapel H, Ogg G. 2011. Direct ex-vivo evaluation of pneumococcal specific T-cells in healthy adults. PLoS One, 6 (10), pp. e25367. | Show Abstract | Read more

Streptococcus pneumoniae is an encapsulated bacterium that causes significant global morbidity and mortality. The nasopharynxes of children are believed to be the natural reservoir of pneumococcus and by adulthood nasopharyngeal carriage is infrequent; such infrequency may be due to demonstrable pneumococcal specific T and B-cell responses. HLA Class 2 tetrameric complexes have been used to characterise antigen specific T-cell responses in a variety of models of infection. We therefore sought to determine the frequency and phenotype of pneumococcal specific T-cells, using a novel HLA-DRB1*1501 tetramer complex incorporating a recently defined T-cell epitope derived from the conserved pneumococcal serine/threonine kinase (StkP). We were able to detect direct ex-vivo StkP(446-60)-tetramer binding in HLA-DRB1*1501 adults. These StkP(446-60)-tetramer binding T-cells had increased CD38 expression and were enriched in CCR7- CD45RA+ expression indicating recent and on-going activation and differentiation. Furthermore, these StkP(446-60)-tetramer binding T-cells demonstrated rapid effector function by secreting interferon-gamma on stimulation with recombinant StkP. This is the first study to directly enumerate and characterise pneumococcal specific T-cells using HLA class 2 tetrameric complexes. We found that ex-vivo pneumococcal-specific T cells were detectable in healthy adults and that they were enriched with cell surface markers associated with recent antigen exposure and later stages of antigen-driven differentiation. It is likely that these activated pneumococcal specific T-cells reflect recent immunostimulatory pneumococcal exposure in the nasopharynx and it is possible that they may be preventing subsequent colonisation and disease.

Malavige GN, Rostron T, Rohanachandra LT, Jayaratne SD, Fernando N, De Silva AD, Liyanage M, Ogg G. 2011. HLA class I and class II associations in dengue viral infections in a Sri Lankan population. PLoS One, 6 (6), pp. e20581. | Show Abstract | Read more

BACKGROUND: HLA class I and class II alleles have been shown to be associated with the development of dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) in different populations. However, the majority of studies have been based on limited numbers of patients. In this study we aimed to investigate the HLA-class I and class II alleles that are positively and negatively associated with the development of DSS in a cohort of patients with DHF and also the alleles associated with development of DHF during primary dengue infections in a Sri Lankan population. METHODOLOGY/PRINCIPAL FINDINGS: The allele frequencies of HLA class I and class II alleles were compared in 110 patients with DHF and 119 individuals from the population who had never reported a symptomatic dengue infection at the time of recruitment. We found that HLA-A*31 (corrected P = 0.01) and DRB1*08 (corrected P = 0.009) were associated with susceptibility to DSS when infected with the dengue virus, during secondary dengue infection. The frequency of DRB1*08 allele was 28.7 times higher than in the normal population in patients with DSS. HLA-A*31 allele was increased 16.6 fold in DHF who developed shock when compared to those who did not develop shock. A*24 (corrected P = 0.03) and DRB1*12 (corrected P = 0.041) were strongly associated with the development of DHF during primary dengue infection. CONCLUSIONS/SIGNIFICANCE: These data suggest that certain HLA alleles confer susceptibility/protection to severe dengue infections. As T cell epitope recognition depend on the HLA type of an individual, it would be now important to investigate how epitope specific T cells associate with primary and secondary dengue infections and in severe dengue infections.

Cited:

45

Scopus

Gutowska-Owsiak D, Schaupp AL, Salimi M, Taylor S, Ogg GS. 2011. Interleukin-22 downregulates filaggrin expression and affects expression of profilaggrin processing enzymes British Journal of Dermatology, 165 (3), pp. 492-498. | Show Abstract | Read more

Summary Background The identification of filaggrin mutations has contributed towards our understanding of hereditary factors associated with epidermal dysfunction observed in individuals with atopic eczema (AE). However, factors that predispose to acquired filaggrin modulation are not well understood. Interleukin (IL)-22 is upregulated in lesional AE tissue, but its effects on filaggrin expression and genes associated with epidermal function have not yet been comprehensively addressed. Objectives To investigate the effects of IL-22 on expression of filaggrin and genes encoding proteins relevant to epidermal function. Methods Microarray analysis was performed on IL-22-stimulated HaCaT keratinocytes. Filaggrin protein level was assessed by an intracellular enzyme-linked immunosorbent assay (ELISA) and Western blot in HaCaT cells and the findings were validated in primary keratinocytes. Results Exposure to IL-22 cytokine resulted in a downregulation of profilaggrin mRNA expression in HaCaT keratinocytes. The expression of genes involved in enzymatic processing of profilaggrin as well as the generation of natural moisturizing factor was also altered. Furthermore, there was an upregulation of many transcripts encoding proteins of the S100 family. Profilaggrin/filaggrin downregulation was detected by intracellular ELISA and Western blot in HaCaT cells. The relevance to the primary setting was confirmed in primary keratinocytes by Western blot. Conclusions IL-22 downregulates profilaggrin/filaggrin expression in keratinocytes at both mRNA and protein levels and affects genes relevant to epidermal function. This novel pathway may have relevance to the pathogenesis and treatment of atopic and other skin disease. © 2011 British Association of Dermatologists.

Crack LR, Chan HW, McPherson T, Ogg GS. 2011. Phenotypic analysis of perennial airborne allergen-specific CD4 + T cells in atopic and non-atopic individuals Clinical and Experimental Allergy, 41 (11), pp. 1555-1567. | Show Abstract | Read more

Background Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4 + T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. Objective To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4 + T cells in adult individuals with severe persistent AD in comparison with healthy controls. Methods Using HLA class II tetrameric complexes based on a HLA-DPB1 *0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Results Ex vivo Fel d 1-specific DPB1 *0401-restricted CD4 + T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Conclusions and Clinical Relevance Circulating Fel d 1-specific DPB1 *0401-restricted CD4 + T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. © 2011 Blackwell Publishing Ltd.

Gomes PL, Malavige GN, Fernando N, Mahendra MH, Kamaladasa SD, Seneviratne JK, Karunatilaka DH, Ogg GS. 2011. Characteristics of Staphylococcus aureus colonization in patients with atopic dermatitis in Sri Lanka. Clin Exp Dermatol, 36 (2), pp. 195-200. | Show Abstract | Read more

BACKGROUND: Colonization of the skin of patients with atopic dermatitis (AD) by Staphylococcus aureus (SA) is associated with more severe disease. AIM: To determine the association of SA colonization patterns and densities in lesional and nonlesional skin in patients with varying severities of AD, and to determine the antibiotic sensitivity patterns of SA isolates from Sri Lanka. METHODS: Skin and nasal swabs collected from 100 patients with AD and 120 controls were used to investigate the presence of SA. Severity of AD was graded using the Nottingham Eczema Severity Score. Colony counts were obtained for skin samples, and antibiotic sensitivity testing was performed in cases positive for SA. RESULTS: Skin colonization was seen in 57 patients (57%) but in only 10 controls (8%). Lesional skin of most patients (52/57; 91%) had SA densities of > 300 colony-forming units/cm(2) . Colonization rates with SA significantly increased with increasing age (Spearman correlation coefficient R = 0.9, P < 0.05) and increasing duration of lesions in patients with AD (Spearman R = 0.87, P < 0.05). Isolates from eight patients (13.5%) were found to be methicillin-resistant S. aureus (MRSA). Only 6 isolates (10%) were susceptible to penicillin and 22 (37%) to erythromycin, while 28 (47%) isolates had erythromycin-induced resistance to clindamycin. CONCLUSIONS: SA colonization rates were significantly associated with increasing age and severity of AD, and particularly with duration of lesions. Patients with severe disease were also more likely to be colonized with SA strains resistant to conventional antibiotics.

Aslam A, Lloyd-Lavery A, Warrell DA, Misbah S, Ogg GS. 2011. Common filaggrin null alleles are not associated with hymenoptera venom allergy in Europeans. Int Arch Allergy Immunol, 154 (4), pp. 353-355. | Show Abstract | Read more

The association of filaggrin mutations with atopic eczema (atopic dermatitis, AD) is well established and it is thought that filaggrin dysfunction impairs the skin's barrier function allowing allergen penetration and subsequent cutaneous sensitisation and inflammation. However, as most forms of barrier dysfunction are not associated with allergic sensitisation to common allergens, the possibility that filaggrin itself is involved in Th1/Th2 polarisation remains. We tested the hypothesis that allergen delivered to the skin independently of the stratum corneum is not associated with filaggrin mutations. Wasp stings bypass the stratum corneum and deliver antigen to the dermis. We found that European individuals with AD (n = 32) have an increased frequency of the 2 commonest filaggrin null mutations (R501X and 2282del4) compared to those with vespid allergy (n = 56) and healthy controls (n = 30). Thus, filaggrin does not appear to have a downstream effect on the development of allergic disease, and it is indeed filaggrin's role in the epithelial function that is likely to determine the link between filaggrin mutations and allergic sensitisation.

Aslam A, Mason A, Zemenides S, Chan H, Nováková L, Branny P, Finn A, Chapel H, Ogg GS. 2010. Rapid effector function of circulating CD4+ T cells specific for immunodominant regions of the conserved serine/threonine kinase found in Streptococcus pneumoniae (StkP) in healthy adults. FEMS Immunol Med Microbiol, 60 (2), pp. 113-122. | Show Abstract | Read more

Streptococcus pneumoniae is an encapsulated bacterium that causes significant global morbidity and mortality. There is emerging evidence that T cells contribute to the immunity that protects humans from S. pneumoniae-associated disease. However, no T-cell epitopes have been identified as yet in this bacterium and there are no data that address the functional nature of T cells specific for pneumococcal-derived epitopes. We sought to define T-cell epitopes in the conserved serine/threonine kinase, found in S. pneumoniae (StkP) and to investigate specific interferon γ (IFN-γ) production resulting from such T-cell activation in healthy donors. We were able to detect the activation of T cells in response to pneumococcal whole-cell antigen or StkP-derived peptides in all 15 individuals. We found that the majority of the T-cell responses were directed against the extracellular, penicillin-binding protein and serine/threonine kinase-associated domains. We proceeded to characterize the immunodominant epitope in detail and observed HLA-DRB1(*) 1501 restriction. This is the first study that has identified T-cell responses to peptides derived from a protein from S. pneumoniae and has shown that in healthy adults, specific T cells have rapid IFN-γ production compatible with effector cell differentiation. The use of such T-cell epitopes will aid in the future monitoring of T-cell responses to both S. pneumoniae infection and vaccination in humans.

Malavige GN, Jones L, Kamaladasa SD, Wijewickrama A, Seneviratne SL, Black AP, Ogg GS. 2010. Natural Killer Cells During Primary Varicella Zoster Virus Infection. J Infect, 61 (2), pp. 190-192. | Read more

McPherson T, Sherman VJ, Aslam A, Crack L, Chan H, Lloyd-Lavery A, Jones L, Ardern-Jones M, Ogg G. 2010. Filaggrin null mutations associate with increased frequencies of allergen-specific CD4+ T-helper 2 cells in patients with atopic eczema. Br J Dermatol, 163 (3), pp. 544-549. | Show Abstract | Read more

BACKGROUND: Filaggrin null mutations associate with atopic eczema and also with asthma when present with eczema. However, while epidermal dysfunction is an important factor in disease pathogenesis, it is unclear how such dysfunction interacts with immune responses to contribute to cutaneous and other inflammatory atopic disease. OBJECTIVES: To gain a better understanding of the mechanisms underlying such predisposition in order to understand different disease phenotypes and possibly identify potential treatment targets. METHODS: We studied 33 individuals with atopic eczema and used interleukin-4 immunospot and human leucocyte antigen class II tetrameric complexes to investigate the peripheral blood allergen-specific CD4+ T-cell responses. RESULTS: Filaggrin null mutations associated with significantly (P<0·05) higher frequencies of allergen-specific CD4+ T-helper 2 cell responses. CONCLUSIONS: These data would support a model where barrier dysfunction possibly promotes greater allergen penetration and delivery to drive allergen-specific CD4+ T cells. This could further contribute to respiratory and cutaneous inflammatory disease.

McPherson T, Aslam A, Crack L, Chan H, Jones L, Ogg G. 2010. Frequencies of circulating allergen-specific T cells temporally associate with longitudinal changes in severity of cutaneous atopic disease. Clin Exp Dermatol, 35 (7), pp. 786-788. | Show Abstract | Read more

Increased levels of allergen-specific T-cells have been documented in the peripheral blood of patients with atopic dermatitis (AD) compared with nonatopic controls. However, little is known about how these relate to disease severity. This study sought to examine if frequencies of circulating allergen-specific T cells correlate with changes in clinical disease severity in a cohort of seven adults with AD who were positive for human leucocyte antigen DRB1*1501. We found that frequencies of allergen-specific CD4+ T cells across the study group were not significantly (P > 0.05) associated with clinical disease severity; however, longitudinal changes within an individual did correlate significantly (P < 0.01) with changes in disease severity. These findings support a role for allergen-specific T-cells in disease pathogenesis.

Baldo M, Bailey A, Bhogal B, Groves RW, Ogg G, Wojnarowska F. 2010. T cells reactive with the NC16A domain of BP180 are present in vulval lichen sclerosus and lichen planus. J Eur Acad Dermatol Venereol, 24 (2), pp. 186-190. | Show Abstract | Read more

BACKGROUND: Lichen sclerosus (LS) is a chronic inflammatory skin condition. The recent demonstration of circulating autoantibodies to extracellular matrix protein 1 and to basement membrane zone (BMZ) components, chiefly BP180, suggests that autoimmunity to these components might contribute to pathogenesis. However, there is no binding of autoantibodies in vivo and as LS is characterized by a lymphocytic infiltrate, it seems likely that LS is mediated, in part, by antigen-specific lymphocytes. Similar mechanisms may apply to vulval lichen planus (LP), an interface dermatitis, with clinical and immunological overlap with LS. OBJECTIVES: This study aims to test the hypothesis that T cells reactive with the NC16A domain of BP180 are present in the peripheral blood of patients with vulval LS and LP. METHODS: Isolated peripheral blood mononuclear cells from 14 patients with vulval LS, 5 with vulval LP and 4 healthy controls were grown in vitro. We examined for immunogenicity of overlapping peptides spanning the NC16A domain of BP180 using interferon-gamma enzyme-linked immunospot assay (ELIspot) on the cultured T-cell lines. BMZ antibodies were assayed, HLA type determined and clinical parameters noted. RESULTS: Significant interferon-gamma production was observed in response to the NC16A peptides in 6 of the 14 vulval LS and 2 of the 5 LP patients, but not in the control subjects. There was an associated autoantibody response to BP180 in 3 LS and 1 LP patient with T-cell responses. These data suggest that in some vulval LS and LP patients, NC16A domain-specific T cells circulate at sufficiently high frequency to be detectable in vitro and show rapid effector function. There was no association with HLA type or clinical parameters. CONCLUSION: We have demonstrated that in > 40% of our vulval LS and LP patients, the NC16A domain of BP180 is a target for circulating T cells, and in vulval LS and LP there are associated autoantibodies to BP180.

Malavige GN, Rohanachandra LT, Jones L, Crack L, Perera M, Fernando N, Guruge D, Ogg GS. 2010. IE63-specific T-cell responses associate with control of subclinical varicella zoster virus reactivation in individuals with malignancies. Br J Cancer, 102 (4), pp. 727-730. | Show Abstract | Read more

BACKGROUND: Reactivation of the varicella zoster virus (VZV) is more common in patients with malignancies; however, the molecular and cellular mechanisms underlying this susceptibility are unclear. METHODS: Using ex vivo interferon-gamma ELISpot assays, we set out to analyse VZV-specific immune responses in a large cohort of patients with malignancies. RESULTS: We observed that patients with malignancies had impaired VZV-specific T-cell responses, particularly in those with haematological malignancies and breast carcinoma. Immediate-early protein 63 (IE63)-specific T-cell responses were significantly impaired in those with subclinical VZV re-activation. CONCLUSIONS: Our results suggest that T-cell responses to IE63 are important in controlling VZV replication.

Aslam A, Chan H, Ogg GS, Warrell DA, Misbah S. 2010. Tracking Antigen-Specific T-Cells during Clinical Tolerance Induction in Humans PLoS ONE, 5 (6), | Show Abstract | Read more

Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigenspecific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen specific T-cell responses during immunotherapy can provide novel insights into mechanisms of tolerance induction in humans and identify new potential treatment targets. © 2010 Aslam et al.

Aslam A, Chan H, Warrell DA, Misbah S, Ogg GS. 2010. Tracking antigen-specific T-cells during clinical tolerance induction in humans. PLoS One, 5 (6), pp. e11028. | Show Abstract | Read more

Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen specific T-cell responses during immunotherapy can provide novel insights into mechanisms of tolerance induction in humans and identify new potential treatment targets.

Jones L, Malavige G, Jeffery K, Kemp E, Breuer J, Klenerman P, Ogg GS. 2009. Tracking epitope-specific antiviral CD4+ T cell responses to a live attenuated vaccine reveals ongoing functional responses. Vaccine, 27 (52), pp. 7398-7401. | Show Abstract | Read more

There are few studies that have examined the frequencies of epitope-specific CD4(+) T cells following the use of a highly effective vaccine, yet such data would potentially be of value for the development of novel vaccination strategies. In this study we tracked human epitope-specific CD4(+) T cell responses over time after immunisation with a live attenuated varicella zoster virus vaccine by MHC Class II tetrameric complexes and functional assays. We show that the peptide-specific responses reflect those against whole virus antigens, and are similar in both frequency and phenotype to those found in healthy volunteers, despite a highly attenuated and clinically inapparent infection.

McPherson T, Ogg G. 2009. Spontaneous resolution of basal cell carcinoma in naevoid basal cell carcinoma syndrome/Gorlin's syndrome. Clin Exp Dermatol, 34 (8), pp. e884-e885. | Show Abstract | Read more

We describe a 10-year-old patient with naevoid basal cell carcinoma syndrome (NBCCS) which was diagnosed when she was 3 years old. She has developed multiple basal cell carcinomas (BCCs) over this time, in particular on her face and trunk. However, we are interested to report that at least two have resolved spontaneously over 2 years without any treatment. This phenomenon has not previously been reported and we believe that it could be important for understanding lesional biology and for future approaches to management.

Horlock C, Stott B, Dyson J, Ogg G, McPherson T, Jones L, Sewell AK, Wooldridge L, Cole DK, Stebbing J, Savage P. 2009. ELISPOT and functional T cell analyses using HLA mono-specific target cells. J Immunol Methods, 350 (1-2), pp. 150-160. | Show Abstract | Read more

Simple T cell assays specific for any chosen HLA class I or class II/peptide combination, are of enormous value in cancer immunotherapy, clinical trials, vaccine and infectious disease research. The reliable measurement of T cell activity can be difficult due to the presence of other alleles on target cells, particularly for the non-HLA-A2 alleles, and the varying baseline characteristics of the different APCs employed. In the absence of pulsing with HLA-A2 restricted peptides, T2 cells are functionally HLA class I and II negative. By coating these cells with recombinant HLA peptide complexes, HLA mono-specific cells are produced that present only a defined single epitope, and generate minimal background immune activation. In ELISPOT, intracellular cytokine staining (ICS) and killing assays using T cells specific for HLA-A2/peptide complexes, the HLA mono-specific cells gave comparable results, to those using standard peptide pulsed HLA-A2 positive T2 cells without significant background. Successful T cell assays for non-HLA-A2 T cells were also performed, with PBMCs recognizing HLA-A24 and HLA-DR15/peptide complexes. The data, obtained with ELISPOT, ICS and FACS-based killing assays, all demonstrate high specificity of T cell activity and low levels of background activity. HLA mono-specific cells are simple to prepare, and can be used with any stable recombinant HLA allele/peptide combination; providing a useful system for improved T cell functional analyses across all HLA allotypes. This represents a significant advance in the generation of reliable functional T cell data.

Black AP, Jones L, Malavige GN, Ogg GS. 2009. Immune evasion during varicella zoster virus infection of keratinocytes. Clin Exp Dermatol, 34 (8), pp. e941-e944. | Show Abstract | Read more

T cells are sensitized during varicella-zoster virus (VZV) infection and are important for control of viral spread and reactivation. In this report, we show that human keratinocytes infected with VZV inhibited upregulation of major histocompatibility complex (MHC) class I, MHC class II and intercellular adhesion molecule-1 after interferon (IFN)-gamma treatment. The ability of keratinocytes to upregulate MHC class I in response to IFN-alpha, tumour necrosis factor (TNF)-alpha and Toll-like receptor (TLR)-3 ligand was also diminished upon VZV infection. VZV-infected keratinocytes treated with IFN-gamma had significantly reduced capacity to stimulate antigen-specific T cells compared with uninfected cells. Interference with IFN-alpha, TNF-alpha, IFN-gamma and TLR-3 signalling in keratinocytes by VZV may contribute to immune evasion of the adaptive immune response.

Ogg G. 2009. Role of T cells in the pathogenesis of atopic dermatitis. Clin Exp Allergy, 39 (3), pp. 310-316. | Show Abstract | Read more

Our understanding of the pathogenesis of atopic dermatitis has been dramatically enhanced following the identification of the association with loss of function mutations in filaggrin, an epidermal protein thought to be important for cutaneous barrier integrity. However, it has also emerged that Th2 cytokines can influence barrier function, including through modulation of the expression of filaggrin, other structural proteins and peptides important for microbial barrier function. A picture is developing of a complex interplay between epidermal and non-epidermal susceptibilities that contribute to disease. Understanding the key components involved will be important for the identification of new approaches to treatment.

Bateman EA, Ardern-Jones MR, Ogg GS. 2008. Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes. Clin Exp Allergy, 38 (11), pp. 1760-1768. | Show Abstract | Read more

BACKGROUND: Characterization of T cell epitopes restricted by common HLA alleles is a powerful tool in the understanding of the immune responses to allergens and for the identification of potential peptides for future peptide immunotherapy (PIT). One important requirement is the identification and use of peptides that will bind to HLA molecules covering a large proportion of the population. OBJECTIVE: To identify commonly recognized CD4(+) T cell epitopes in Fel d 1, restricted through frequently expressed HLA molecules for potential future use in PIT. METHODS: HLA matched antigen presenting cells, HLA blocking antibodies, and peptide truncations were used in ELISpot assays to establish HLA-restricted T cell epitopes. Cytokine responses were measured by ex vivo and cultured IFN-gamma, IL-4, and IL-10 ELISpots. RESULTS: Responses to an immunodominant region of chain 2 were identified in the majority of atopic individuals and epitopes restricted by HLA-DQB1(*)06 and -DPB1(*)0401 were characterized in detail. Significantly higher ex vivo IL-4 and lower IFN-gamma responses were observed to both epitopes in individuals with atopic dermatitis (AD) compared with those without disease. IL-10 responses were significantly lower in those with AD in the individuals with HLA-DPB1(*)0401. CONCLUSIONS: We have identified an immunodominant region of Fel d 1 which is frequently recognized by CD4(+) T cells from atopic individuals and contains epitopes that are restricted by very common HLA alleles.

Malavige GN, Jones L, Black AP, Ogg GS. 2008. Varicella zoster virus glycoprotein E-specific CD4+ T cells show evidence of recent activation and effector differentiation, consistent with frequent exposure to replicative cycle antigens in healthy immune donors. Clin Exp Immunol, 152 (3), pp. 522-531. | Show Abstract | Read more

Varicella zoster viru (VZV)-specific T cell responses are believed to be vital in recovery from primary VZV infection and also in the prevention of viral reactivation. While glycoprotein E (gE) is the most abundant and one of the most immunogenic proteins of the virus, there are no data addressing potential T cell epitopes within gE, nor the phenotype of specific T cells. Using interferon gamma enzyme-linked immunospot assays and intracellular cytokine assays, we identified gE-specific immune responses in 20 adult healthy immune donors which were found to be dominated by the CD4+ subset of T cells. We characterized three immune dominant epitopes within gE restricted through DRB1*1501, DRB1*07 and DRB4*01, and used DRB1*1501 class II tetrameric complexes to determine the ex vivo frequency and phenotype of specific T cells. In healthy immune donors, the cells were largely positive for CCR7, CD28 and CD27, but expressed variable CD62L and low levels of cutaneous lymphocyte associated antigen with evidence of recent activation. In summary, we show that circulating gE-specific CD4+ T cells are detected at a relatively high frequency in healthy immune donors and show evidence of recent activation and mixed central and effector memory phenotype. These data would be compatible with frequent exposure to replicative cycle antigens in healthy donors and are consistent with a role for gE-specific CD4+ T cells in the control of viral replication.

Jones L, Black A, Malavige G, Ogg G. 2008. Phenotypic analysis of human CD4+T cells specific for IE63 protein of VZV JOURNAL OF INFECTION, 56 (4), pp. 300-300. | Read more

Malavige G, Jones L, Black A, Wijewickrama A, Seneviratne S, Kamaladasa S, Ogg G. 2008. Viral loads in primary varicella zoster virus infection JOURNAL OF INFECTION, 56 (4), pp. 307-307. | Read more

Ardern-Jones MR, Black AP, Ogg GS. 2008. Anti-lymphocyte function associated antigen-1 inhibits T-helper 2 function of human allergen-specific CD4+ T cells. Br J Dermatol, 158 (3), pp. 456-462. | Show Abstract | Read more

BACKGROUND: Blockade of lymphocyte function associated antigen-1 (LFA-1) is proving successful in the management of psoriasis and other inflammatory skin conditions including atopic dermatitis (AD), but the dependence of allergen-specific CD4+ T-cell function on LFA-1 has not been studied extensively. OBJECTIVES: We sought to investigate the potential ability of LFA-1 inhibition to influence keratinocyte presentation of allergen to specific T-helper (Th) 2 cell clones. METHODS: Using human leucocyte antigen class II tetrameric complexes, we generated Der p 1-specific DRB1*1501-restricted CD4+ T-cell lines (n=5) and clones (n=4) from the peripheral blood of five adults with AD. RESULTS: Using doses of anti-LFA-1 present in vivo, we observed significant inhibition (P<0.05) of allergen-specific CD4+ T-cell production of interleukin-4 with such inhibition occurring during presentation of allergen by keratinocytes. CONCLUSIONS: These data show that at doses present in vivo, LFA-1 blockade inhibits keratinocyte presentation to allergen-specific Th2 cells, suggesting one mechanism through which anti-LFA-1 may be beneficial therapeutically.

Malavige GN, Jones L, Kamaladasa SD, Wijewickrama A, Seneviratne SL, Black AP, Ogg GS. 2008. Viral load, clinical disease severity and cellular immune responses in primary varicella zoster virus infection in Sri Lanka. PLoS One, 3 (11), pp. e3789. | Show Abstract | Read more

BACKGROUND: In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied. METHODOLOGY: We used IFNgamma ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection. PRINCIPAL FINDINGS: Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNgamma ELISpot responses of patients (P<0.001, r = -0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation. CONCLUSIONS: Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses.

Jones L, Black AP, Malavige GN, Ogg GS. 2007. Phenotypic analysis of human CD4+ T cells specific for immediate-early 63 protein of varicella-zoster virus. Eur J Immunol, 37 (12), pp. 3393-3403. | Show Abstract | Read more

Open reading frame 63 of varicella-zoster Virus (VZV) encodes an immediate early (IE) phosphoprotein (IE63) that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication; however, data addressing the existence of IE63 protein-specific CD4+ T cells are limited. Using IFN-gamma immunosorbent assays, we identified high frequencies of responses to overlapping peptides spanning the IE63 protein both ex vivo and after in vitro restimulation in healthy VZV-seropositive individuals. We identified a commonly recognised epitope, restricted by HLA-DRB1*1501, which was naturally processed and presented by keratinocytes. We proceeded to investigate the frequency and phenotype of the epitope-specific CD4+ T cells using HLA class II tetrameric complexes. Epitope-specific CD4+ T cells were detectable ex vivo and showed a mixed central and effector-memory differentiation phenotype, with a significant proportion showing evidence of recent activation and rapid effector function. In summary these data implicate persistent low-level or recurrent VZV antigen exposure in healthy immune donors and are compatible with a role for IE63-specific CD4+ T cells in the control of viral reactivation.

Malavige GN, Rostron T, Seneviratne SL, Fernando S, Sivayogan S, Wijewickrama A, Ogg GS. 2007. HLA analysis of Sri Lankan Sinhalese predicts North Indian origin. Int J Immunogenet, 34 (5), pp. 313-315. | Show Abstract | Read more

The origin of the Sinhalese population of Sri Lanka is debated. We subtyped HLA-A*02 in 101 Sinhalese and observed a preponderance of the rare allele HLA-A*0211 which was similar to reported frequencies in northern India. Taken with low-resolution typing for the remaining A, B, C, DR and DQ alleles, these data suggest a North Indian origin for the Sri Lankan Sinhalese.

Bateman EA, Ardern-Jones M, Ogg GS. 2007. Dose-related reduction in allergen-specific T cells associates with clinical response of atopic dermatitis to methotrexate. Br J Dermatol, 156 (6), pp. 1376-1377. | Read more

Black AP, Ardern-Jones MR, Kasprowicz V, Bowness P, Jones L, Bailey AS, Ogg GS. 2007. Human keratinocyte induction of rapid effector function in antigen-specific memory CD4+ and CD8+ T cells. Eur J Immunol, 37 (6), pp. 1485-1493. | Show Abstract | Read more

The ability of human keratinocytes to present antigen to T cells is controversial and, indeed, it has been suggested that keratinocytes may promote T cell hyporesponsiveness. Furthermore, it is unclear whether keratinocytes can process antigen prior to MHC class I and class II presentation. We tested the ability of keratinocytes to induce functional responses in epitope-specific CD4+ and CD8+ memory T cells using peptides, protein and recombinant expression vectors as sources of antigen. Keratinocytes were able to efficiently process and present protein antigen to CD4+ T cells, resulting in cytokine secretion (Th1 and Th2). This interaction was dependent on keratinocyte expression of HLA class II and ICAM-1, which could be induced by IFN-gamma. In addition, keratinocytes could present virally encoded or exogenous peptide to CD8+ T cells, resulting in T cell cytokine production and target cell lysis. Finally, T cell lines grown using keratinocytes as stimulators showed no loss of function. These findings demonstrate that keratinocytes are able to efficiently process and present antigen to CD4+ and CD8+ memory T cells and induce functional responses. The findings have broad implications for the pathogenesis of cutaneous disease and for transcutaneous drug or vaccine delivery.

Ardern-Jones MR, Black AP, Bateman EA, Ogg GS. 2007. Bacterial superantigen facilitates epithelial presentation of allergen to T helper 2 cells. Proc Natl Acad Sci U S A, 104 (13), pp. 5557-5562. | Show Abstract | Read more

Although clinical and laboratory evidence support roles for both staphylococcal infection and environmental allergens in the pathogenesis of atopic dermatitis, human studies have largely considered these variables independently. We sought to test the hypothesis that staphylococcal superantigen influences the allergen-specific T cell response. We first mapped a Der p 1 epitope and used HLA DRB1*1501 class II tetramer-based cell sorted populations to show that specific CD4(+) T cells were able to recognize the peptide presented by HLA DR-matched keratinocytes. We observed that staphylococcal enterotoxin B (SEB) enhanced the IL-4 Der p 1-specific T cell response. This response was mediated by two synergistic mechanisms: first, SEB-induced IFN-gamma promoted class II and intercellular adhesion molecule-1 expression by presenting keratinocytes; and second, SEB-induced IL-4 directly amplified allergen-specific CD4(+) T cell production of many cytokines. We propose that handling of staphylococcal infection is a critical step in the amplification of the allergen-specific T cell response, linking two common disease associations and with implications for the prevention and treatment of atopic disease.

Malavige GN, Jones L, Black AP, Ogg GS. 2007. Rapid effector function of varicella-zoster virus glycoprotein I-specific CD4+ T cells many decades after primary infection. J Infect Dis, 195 (5), pp. 660-664. | Show Abstract | Read more

Glycoprotein I (gI) of varicella-zoster virus (VZV) contributes to viral virulence and is therefore a potentially important target for T cell control of viral replication. Persisting effector function of gI-specific T cells after primary infection has not been previously examined. We have shown that, many decades after infection, relatively high frequencies gI-specific interferon- gamma responses are detectable ex vivo and are dominated by CD4(+) T cells. We characterized the optimal peptide of the strongest response in our cohort showing restriction through DRB4*01. These findings are consistent with gI-specific CD4(+) T cell involvement in the control of VZV replication.

Savage P, Dyson J, Milrain M, Mathews D, King B, Chan HT, Barber L, Epenetos A et al. 2007. Immunotherapy with antibody-targeted HLA class I complexes: results of in vivo tumour cell killing and therapeutic vaccination. Tumour Biol, 28 (4), pp. 205-211. | Show Abstract | Read more

BACKGROUND: The delivery of antibody-targeted major histocompatibility complex (MHC) class I complexes containing immunogenic peptides to the surface of tumour cells allows cytotoxic T lymphocytes (CTLs) of non-tumour specificity to recognise and kill the tumour cell. Previous studies have demonstrated the activity of this system in vitro and in a simple pre-clinical model. This system has also been shown to be an effective method of expanding antigen-specific CTLs in vitro when used to target MHC class I complexes to the surface of B cells. METHODS: Mice were immunised with ovalbumin and the survival of EL4Hu20 lymphoma cells targeted with H2-D(b)/Ova complexes and control MHC complexes was compared by FACS analysis. A tumour protection assay was performed where immunised mice were injected B16Hu20 melanoma cells targeted with H2-K(b)/Ova or control complexes. T cell expansion in vivo was examined by administering B cells targeted with MHC class I/peptide complexes and assessing T cell expansion by tetramer analysis. RESULTS: In vivo killing of H2-D(b)/Ova-targeted lymphoma cells in the immunised mice was demonstrated with these cells present at only 12% of the level of the control cells. In contrast, in non-immunised mice the survival of H2-D(b)/Ova-targeted and control cells was comparable. In the tumour protection assay, injection of melanoma cells targeted with H2-K(b)/Ova complexes resulted in the development of only a solitary metastasis in each mouse. This compared to an average of 130 metastases in the control mice injected with B16Hu20 cells targeted with a control MHC peptide complex. In vivo CTL expansion was demonstrated after a single intravenous administration of Daudi B cells coated with H2-D(b)/Uty complexes produced an increase in the proportion of Uty-reactive CTLs from 3.3 to 21.5%. CONCLUSION: This study supports the development of antibody-delivered MHC complexes as a method of producing CTL-mediated lysis of cancer cells in vivo. As a therapeutic vaccine, the system may provide an effective approach for expanding oligoclonal T cell responses in vivo in the treatment of malignancy and infectious diseases.

Seneviratne SL, Black AP, Jones L, di Gleria K, Bailey AS, Ogg GS. 2007. Interleukin-4 promotes human CD8 T cell expression of CCR7. Immunology, 120 (1), pp. 66-72. | Show Abstract | Read more

Despite strong evidence supporting a pathway of human T cell differentiation characterized by changes in the expression of CCR7, CD28, CD27 and CD62L, few studies have addressed the mechanisms of pathway regulation. Cutaneous lymphocyte-associated antigen (CLA)-positive skin-homing CD8(+) T cells expressed significantly elevated levels of activation markers compared with CLA(-) CD8(+) T cells in individuals (n = 27) with cutaneous atopic disease. Despite such an activated phenotype, CLA(+) T cells expressed significantly higher levels of CCR7 than a CLA(-) T cell subset. Interleukin (IL)-4 was found to dramatically promote CCR7 expression by antigen-specific CD8(+) cells. Furthermore, skin-homing CD8(+) T cells from individuals with severe disease produced significantly less IL-10 than those derived from mildly affected atopic subjects. Thus in a T-helper 2 dominated disease, tissue-specific CD8(+) T cells show altered CCR7 expression and cytokine production, which may contribute to continued lymph node homing, antigen presentation and disease. IL-4 promotes expression of CCR7, a marker linked to existing models of CD8(+) T cell differentiation.

Seneviratne SL, Black AP, Jones L, Bailey AS, Ogg GS. 2007. The role of skin-homing T cells in extrinsic atopic dermatitis. QJM, 100 (1), pp. 19-27. | Show Abstract | Read more

BACKGROUND: T cells that express Cutaneous Lymphocyte-Associated antigen (CLA) have the potential of migrating to the skin, and are hypothesized to play a role in cutaneous atopic disease. AIM: To investigate the immune phenotype and cytokine responses to Der p 1 stimulation of CLA+ T cells in extrinsic atopic dermatitis (EAD). DESIGN: In vitro testing, with controls. METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from EAD patients (n=27) and non-atopic healthy individuals (n=22). Phenotypic analysis of naive, CLA+ and non-CLA+ memory/effector CD4+ and CD8+ T cells used markers of cell activation, differentiation, adhesion, apoptosis and chemokine receptor expression. Cytokine responses in these cells were studied following Der p 1 stimulation. RESULTS: CLA+ T cells from EAD patients expressed significantly higher levels of CD25, HLA-DR, CD38, CD71, CXCR1, CXCR2 and lower levels of bcl2, CCR5, CCR7, CXCR3, and CD62L (p<0.05). DISCUSSION: In EAD patients, CLA+ T cells express increased levels of markers associated with activation, adhesion and apoptosis, show differences in the level of expression of differentiation markers and display a distinct chemokine receptor preference, compared with cells from healthy controls. These data suggest a significant role for CLA+ T cells in the pathogenesis of cutaneous atopic disease.

Bateman EA, Ardern-Jones MR, Ogg GS. 2006. Persistent central memory phenotype of circulating Fel d 1 peptide/DRB1*0101 tetramer-binding CD4+ T cells. J Allergy Clin Immunol, 118 (6), pp. 1350-1356. | Show Abstract | Read more

BACKGROUND: Although substantial evidence suggests that T cells are important in the pathogenesis of atopic dermatitis (AD), little is known of the differentiation status of CD4+ T cells specific for common environmental allergens. OBJECTIVE: To determine the frequency, differentiation phenotype, and function of circulating allergen-specific CD4+ T cells in adult individuals with severe persistent AD and controls. METHODS: Using tetrameric complexes of an HLA DRB1*0101 restricted epitope from Fel d 1, the major IgE-reactive component of cat dander, we studied ex vivo and cultured T-cell frequency and phenotype in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IFN-gamma, IL-4, and IL-10 enzyme linked immuno-spot analysis. RESULTS: Ex vivo Fel d 1-specific DRB1*0101-restricted CD4+ T cells express high levels of CCR7, CD62L, CD27, and CD28 and proportionately low levels of tissue-specific homing receptors and TH1 and TH2 cytokine production, placing the cells largely within the central memory subgroup. CONCLUSION: Circulating Fel d 1-specific DRB1*0101-restricted CD4+ T cells maintain central memory capacity, consistent with a potential to contribute to persisting clinical atopic disease. CLINICAL IMPLICATIONS: Persisting central memory characteristics of allergen-specific CD4+ T cells in individuals with AD may contribute to chronic disease.

Black AP, Jones L, Ardern-Jones M, Ogg GS. 2006. A novel fluorescent sensitive assay for detection of differential T cell mediated lysis of multiple adherent target cells. J Immunol Methods, 316 (1-2), pp. 153-157. | Show Abstract | Read more

There are few studies that have investigated T cell mediated lysis of adherent cells. We have developed a novel, rapid and sensitive fluorescent dye-swap assay that allows efficient detection of adherent target cell lysis. The assay allows simultaneous use of multiple differentially sensitised targets and facilitates concomitant surface or intracellular effector cell phenotypic analysis.

Aslam A, Kessler B, Batycka M, O'Callaghan CA, Misbah SA, Warrell DA, Ogg G. 2006. Defining the T cell antigen proteome of wasp venom. Clin Exp Allergy, 36 (10), pp. 1274-1280. | Show Abstract | Read more

BACKGROUND: While modulation of T cell function is believed to be important in the successful acquisition of clinical tolerance during venom immunotherapy, little is known of the role of wasp venom specific T cell antigens. OBJECTIVE: We sought comprehensively to characterize the T cell proteome for wasp venom to facilitate the future development of T cell-based immunotherapeutic approaches. METHODS: Using peripheral blood mononuclear cells from wasp venom-allergic individuals and IL-4 ELISPOT analysis, we characterized T cell responses to whole venom and gel filtration/ion exchange-fractionated venom. Reactive fractions were purified and identified using highly sensitive electrospray ion-trap mass spectrometry. RESULTS: Wasp venom-allergic individuals have detectable whole wasp venom-specific T cells directly ex vivo, which show rapid IL-4 effector function. T cell responses to gel filtration/ion exchange fractionated venom were dominated by responses to phospholipase A(1), hyaluronidase and antigen 5. CONCLUSION: Although it is likely that there are many T cell antigens within wasp venom, the main responses are to proteins coincident with the known IgE-binding proteins.

Jones L, Black AP, Malavige GN, Ogg GS. 2006. Persistent high frequencies of varicella-zoster virus ORF4 protein-specific CD4+ T cells after primary infection. J Virol, 80 (19), pp. 9772-9778. | Show Abstract | Read more

Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-gamma) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-gamma cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.

Seneviratne SL, Jones L, Bailey AS, Black AP, Ogg GS. 2006. Severe atopic dermatitis is associated with a reduced frequency of IL-10 producing allergen-specific CD4+ T cells. Clin Exp Dermatol, 31 (5), pp. 689-694. | Show Abstract | Read more

BACKGROUND: Several studies have investigated levels of T-cell-derived interleukin (IL)-10 in individuals with atopic dermatitis, with conflicting results. AIMS/HYPOTHESIS: In order to address whether stratification of disease severity may help resolve the different findings, the hypothesis was tested that individuals with severe atopic dermatitis have a lower frequency of circulating IL-10-producing, allergen-specific CD4+ T cells than do individuals with mild disease. METHODS: Using peripheral blood mononuclear cells derived from individuals with severe (n=12) and mild atopic dermatitis (n=10) and from nonatopic controls (n=10), we investigated production by CD4+ T cells of tumour necrosis factor (TNF)-alpha, IL-4, IL-5, IL-13 and IL-10 in response to phorbol myristate acetate/ionomycin and Der p1 allergen. RESULTS: It was observed that there were significantly higher frequencies of allergen-specific circulating CD4+ T cells producing TNF-alpha- IL-4-, IL-5- and IL-13, and lower frequencies of these cells producing IL-10 in individuals with severe atopic dermatitis compared with mildly affected individuals and nonatopic controls (P<0.01 for all comparisons). Furthermore, the Der p1-specific CD4+ T cells were enriched within the subset of cells positive for cutaneous lymphocyte-associated antigen. CONCLUSIONS: Analysis of levels of allergen-specific CD4+ T-cell production of IL-10 in relation to disease severity argues in favour of a role for IL-10 in the control of atopic dermatitis.

Rakoff-Nahoum S, Kuebler PJ, Heymann JJ, E Sheehy M, Ortiz GM, S Ogg G, Barbour JD, Lenz J, Steinfeld AD, Nixon DF. 2006. Detection of T lymphocytes specific for human endogenous retrovirus K (HERV-K) in patients with seminoma. AIDS Res Hum Retroviruses, 22 (1), pp. 52-56. | Show Abstract | Read more

Human endogenous retrovirus K (HERV-K) is distinctive among the retroviruses that comprise about 8% of the human genome in that multiple HERV-K proviruses encode full-length viral proteins, and many HERV-K proviruses formed during recent human evolution. HERV-K gag proteins are found in the cytoplasm of primary tumor cells of patients with seminoma. We identified HERV-K-specific T cells in patients with a past history of seminoma using the interferon-gamma ELISPOT assay and an MHC-HERV-K peptide-specific tetramer. A minority of apparently healthy subjects without evident germ cell tumors also made HERV-K-specific T cell responses. In summary, we detected T cell reactivity to HERV-K peptides in both past seminoma patients and a minority of apparently healthy controls.

Ardern-Jones MR, Bateman EA, Black AP, Bailey AS, Ogg GS. 2005. Phenotypic analysis of Der P 1-peptide specific T cells in individuals with severe atopic dermatitis IMMUNOLOGY, 116 pp. 91-91.

Black AP, Bailey A, Jones L, Turner RJ, Hollowood K, Ogg GS. 2005. p53-specific CD8+ T-cell responses in individuals with cutaneous squamous cell carcinoma. Br J Dermatol, 153 (5), pp. 987-991. | Show Abstract | Read more

BACKGROUND: Systemic immunosuppression is a significant risk factor for cutaneous squamous cell carcinoma (SCC). p53 is mutated and overexpressed in up to 90% of cutaneous SCC lesions. Despite considerable evidence that the immune response is important in the control of cutaneous SCC, there are no studies documenting potential tumour-associated antigens. OBJECTIVES: We tested the hypothesis that individuals with cutaneous SCC have functional circulating CD8+ T cells specific for p53. METHODS: Interferon-gamma immunosorbent assays were used to screen peripheral blood mononuclear cells for reactivity to six p53-derived HLA-A*0201-restricted epitopes from HLA-A*0201-positive patients and controls. RESULTS: We observed significantly elevated frequencies of p53-specific CD8+ T cells in seven of 26 individuals with cutaneous SCC and in one of 10 controls. The degree of lymphocytic infiltrate significantly correlated with the frequency of CD8+ T cells specific for p53 epitopes, but not with control epitopes. CONCLUSIONS: Overall, these data suggest that p53 may represent a target for CD8+ T cells in a proportion of individuals with cutaneous SCC.

Seneviratne SL, Jones L, Bailey AS, Samuel RV, Black AP, Ogg GS. 2005. Interleukin-4 induced down-regulation of skin homing receptor expression by human viral-specific CD8 T cells may contribute to atopic risk of cutaneous infection. Clin Exp Immunol, 141 (1), pp. 107-115. | Show Abstract | Read more

Factors controlling the expression of cutaneous lymphocyte-associated antigen (CLA) by T cells are poorly understood, but data from murine and human CD4(+) T cell systems have suggested that cytokines play an important role. However, there are no data examining the influence of cytokines on the expression of CLA by human antigen-specific CD8(+) T cells. Peripheral blood mononuclear cells (PBMC) were isolated from 10 HLA-A*0201-positive healthy individuals. Using HLA-peptide tetrameric complexes refolded with immunodominant peptides from Epstein-Barr virus (EBV), cytomegalovirus (CMV) and influenza A virus, we investigated the temporal associations of CLA expression by viral-specific CD8(+) T cells following stimulation with antigen. Ex vivo influenza matrix-specific CD8(+) T cells expressed significantly (P < 0.05) greater levels of CLA than EBV BMLF1 and CMV pp65-specific CD8(+) T cells (mean 9.7% influenza matrix versus 1.4% BMLF1 versus 1.1% pp65) and these differences were sustained on culture. However, regardless of viral specificity, interleukin (IL)-12 and IL-4 induced significant (P < 0.05) dose-dependent up-regulation and down-regulation of CLA expression, respectively, with IL-4 showing a dominant negative effect. In many cases, IL-4 resulted in complete abrogation of detectable CLA expression by the viral-specific CD8(+) T cells. Overall these data demonstrate that CLA expression by human viral-specific CD8(+) T cells is highly dynamic and that IL-4 causes significant down-regulation. Disorders associated with a type 2 cytokine shift may reduce the efficiency of skin homing by viral-specific CD8(+) T cells. Furthermore, the ability to modify the local and systemic microenvironment may offer novel therapeutic strategies that influence tissue-specific T cell homing.

Black AP, Seneviratne SL, Jones L, King AS, Winsey S, Arsecularatne G, Wojnarowska F, Ogg GS. 2004. Rapid effector function of circulating NC16A-specific T cells in individuals with mucous membrane pemphigoid. Br J Dermatol, 151 (6), pp. 1160-1164. | Show Abstract | Read more

BACKGROUND: Mucous membrane pemphigoid (MMP) is a chronic blistering skin disease frequently associated with circulating autoantibodies directed to a number of antigens including the NC16A region of BP180. NC16A domain-specific T cells have been identified in the blood of individuals with bullous pemphigoid (BP), pemphigoid gestationis and linear IgA disease, but there are no data investigating the potential role for such T cells in the pathogenesis of MMP. OBJECTIVES: To test the hypothesis that NC16A-specific T cells exist in the peripheral blood of individuals with MMP. METHODS: We isolated peripheral blood mononuclear cells from 10 patients with MMP, 17 with BP and 10 healthy controls and examined the immunogenicity of overlapping peptides spanning the NC16A domain using interferon (IFN)-gamma enzyme-linked immunospot assay. RESULTS: Significant IFN-gamma production was observed in response to the NC16A peptides in two of the patients with MMP and two of the patients with BP but in none of the normal controls. These data suggest that in a minority of individuals with MMP, NC16A domain-specific T cells circulate at sufficiently high frequency to be detectable directly ex vivo and to show rapid effector function. CONCLUSIONS: Overall, these findings are the first to examine the potential role for antigen-specific autoreactive T cells in the pathogenesis of MMP, and confirm that in some individuals the NC16A domain may be an important target antigen.

Stebbing J, Gazzard B, Patterson S, Bower M, Nelson M, Epenetos A, Ogg G, Gotch F, Savage P. 2004. Simplified one-step antibody-HLA directed expansion of HIV-specific cytotoxic T lymphocytes: a system suited for use in vivo. AIDS, 18 (15), pp. 2099-2101. | Read more

Ho LP, Urban BC, Jones L, Ogg GS, McMichael AJ. 2004. CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion. J Immunol, 172 (12), pp. 7350-7358. | Show Abstract

Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.

Savage P, Gao L, Vento K, Cowburn P, Man S, Steven N, Ogg G, McMichael A, Epenetos A, Goulmy E, Stauss HJ. 2004. Use of B cell-bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen. Blood, 103 (12), pp. 4613-4615. | Show Abstract | Read more

Recent studies have detected Wilms tumor antigen (WT1)-specific cytotoxic T lymphocytes (CTLs) in patients with acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) and demonstrated that most of these CTLs were low avidity. Although HLA-mismatched donors can mount high-avidity CTLs against HLA-A2-presented peptides of WT1, a dominant anti-alloimmune response usually obscures detection of peptide-specific CTLs. Here we explored the feasibility of using recombinant HLA-A2 monomers containing single peptide epitopes as immunogens to generate peptide-specific CTLs from allogeneic donors. We demonstrate that the coating of HLA-A2(-) B lymphocytes with A2/peptide monomers provides a strong stimulus for autologous peptide-specific CTLs. After 3 to 5 rounds of stimulation a population of CD8(+) T cells binding A2/peptide tetramers is easily detectable by fluorescence-activated cell sorting analysis. Furthermore, sorted A2/WT1 tetramer-positive CTLs display strong cytotoxic activity against leukemia cells expressing WT1 endogenously but not against WT1(-) human tumor cells. Thus, HLA/peptide monomers may be useful to isolate peptide-specific donor lymphocytes for treatment of patients with leukemia after HLA-mismatched transplantation.

Webster GJ, Reignat S, Brown D, Ogg GS, Jones L, Seneviratne SL, Williams R, Dusheiko G, Bertoletti A. 2004. Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol, 78 (11), pp. 5707-5719. | Show Abstract | Read more

The cytotoxic T-cell response in chronic hepatitis B virus (HBV) infection has been described as weak and mono- or oligospecific in comparison to the more robust virus-specific T-cell response present in resolved infection. However, chronic hepatitis B is a heterogeneous disease with markedly variable levels of virus replication and liver disease activity. Here we analyzed (both directly ex vivo and after in vitro stimulation) the HBV-specific CD8 T-cell responses against structural and nonstructural HBV proteins longitudinally in patients with different patterns of chronic infections. We found that the profiles of virus-specific CD8(+)-T-cell responses during chronic infections are highly heterogeneous and influenced more by the level of HBV replication than by the activity of liver disease. An HBV DNA load of <10(7) copies/ml appears to be the threshold below which circulating multispecific HBV-specific CD8(+) T cells are consistently detected. Furthermore, CD8(+) T cells with different specificities are differentially regulated during chronic infections. HBV core-specific CD8(+) T cells are associated with viral control, while CD8(+) T cells specific for envelope and polymerase epitopes can occasionally be found in the setting of high levels (>10(7) copies) of HBV replication. These findings have implications for the design of immunotherapy for chronic HBV infections.

Komai-Koma M, Jones L, Ogg GS, Xu D, Liew FY. 2004. TLR2 is expressed on activated T cells as a costimulatory receptor. Proc Natl Acad Sci U S A, 101 (9), pp. 3029-3034. | Show Abstract | Read more

Toll is the founder of a group of pattern recognition receptors that play a critical role in the innate immunity in Drosophila. At least 10 distinct Toll-like receptors (TLRs), recognizing pathogen-associated molecular patterns, have now been identified in humans. Most investigations on TLRs have focused on cells of the innate system. We report here that naïve human T cells expressed high levels of cell-surface TLR2 after activation by anti-T cell receptor antibody and IFN-alpha. Activated cells produced elevated levels of cytokines in response to the TLR2 ligand, bacterial lipopeptide. Furthermore, CD4(+)CD45RO(+) memory T cells from peripheral blood constitutively expressed TLR2 and produced IFN-gamma in response to bacterial lipopeptide, which also markedly enhanced the proliferation and IFN-gamma production by CD45RO(+) T cells in the presence of IL-2 or IL-15. Thus, TLR2 serves as a costimulatory receptor for antigen-specific T cell development and participates in the maintenance of T cell memory. This suggests that pathogens, via their pathogen-associated molecular patterns, may contribute directly to the perpetuation and activation of long-term T cell memory in both antigen-dependent and independent manner.

Ogg GS. 2003. T-cell immunotherapy of allergic disease: the role of CD8+ T cells. Curr Opin Allergy Clin Immunol, 3 (6), pp. 475-479. | Show Abstract | Read more

PURPOSE OF REVIEW: The scope of this review is to place recent advances in T-cell immunotherapy into an account of our understanding of the potential role of CD8+ T cells in the pathogenesis of allergic disease. RECENT FINDINGS: Studies over the last year suggest that changes in CD8+ T-cell function may represent key events in successful T-cell immunotherapy. The first human human leukocyte antigen class I allergen epitopes have now been described and will provide further insights into the role of allergen-specific CD8+ T cells. SUMMARY: The coupling of recent technical advances in the study of antigen-specific T cells with the knowledge of human allergen class I epitopes will promote rapid progress in the field, with potential consequences for the diagnosis, monitoring and immunotherapeutic treatment of affected individuals.

Black AP, Ogg GS. 2003. The role of p53 in the immunobiology of cutaneous squamous cell carcinoma. Clin Exp Immunol, 132 (3), pp. 379-384. | Show Abstract | Read more

Cutaneous squamous cell carcinoma is typically characterized by the over-expression of the tumour suppressor protein p53. Considerable evidence suggests that immune competence is important in the control of cutaneous SCC. We discuss the immunobiology of p53 and its relevance to cutaneous SCC, including the potential interaction with human papillomavirus.

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.

Papagno L, Appay V, Sutton J, Rostron T, Gillespie GM, Ogg GS, King A, Makadzanhge AT et al. 2002. Comparison between HIV- and CMV-specific T cell responses in long-term HIV infected donors. Clin Exp Immunol, 130 (3), pp. 509-517. | Show Abstract | Read more

The mechanisms underlying non-progression in HIV-1 infection are not well understood; however, this state has been associated previously with strong HIV-1-specific CD8+ T cell responses and the preservation of proliferative CD4+ T cell responses to HIV-1 antigens. Using a combination of interferon-gamma (IFN-gamma) ELISpot assays and tetramer staining, the HIV-1-specific CD8+ T cell populations were quantified and characterized in untreated long-term HIV-1-infected non-progressors and individuals with slowly progressive disease, both in relation to CD4+ T cell responses, and in comparison with responses to cytomegalovirus (CMV) antigens. High levels of CD8+ T cell responses specific for HIV-1 or CMV were observed, but neither their frequency nor their phenotype seemed to differ between the two patient groups. Moreover, while CMV-specific CD4+ T cell responses were preserved in these donors, IFN-gamma release by HIV-1-specific CD4+ T cells was generally low. These data raise questions with regard to the role played by CD8+ T cells in the establishment and maintenance of long-term non-progression.

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Seneviratne SL, Jones L, King AS, Black A, Powell S, McMichael A, Ogg GS. 2002. Allergen-specific CD8(+) T cells and atopic disease JOURNAL OF CLINICAL INVESTIGATION, 110 (9), pp. 1283-1291. | Read more

Seneviratne SL, Jones L, King AS, Black A, Powell S, McMichael AJ, Ogg GS. 2002. Allergen-specific CD8(+) T cells and atopic disease. J Clin Invest, 110 (9), pp. 1283-1291. | Show Abstract | Read more

Considerable evidence suggests that IL-10 may have a role in the manifestation of atopic disease. We sought to test the hypothesis that at the single cell level, allergen-specific T cells have diminished IL-10 production capacity in severely affected atopics compared with asymptomatic atopics. We defined three A*0201-restricted Der p 1 CD8(+) T cell epitopes. Using human leukocyte antigen-A*0201-peptide (HLA-A*0201-peptide) tetrameric complexes and enzyme-linked immunospot assays to analyze peripheral blood mononuclear cells from A*0201-positive severely symptomatic atopics, asymptomatic atopics, and nonatopic controls, we observed a significant association between the frequency of the Der p 1-specific CD8(+) T cells and disease activity. The specific T cells expressed an antigen-experienced cell surface phenotype, and 45.7% were positive for cutaneous lymphocyte-associated antigen. The specific T cells were able to produce IFN-gamma efficiently, but their IL-10 production was significantly reduced in severely affected atopics. In contrast, viral-specific CD8(+) T cells were able to produce equivalent amounts of IL-10 in the severely affected atopics compared with asymptomatic atopics and nonatopics. Through defining the first human atopic allergen HLA class I epitopes, we have provided a possible cellular mechanism to link the previous association of low IL-10 levels and severe atopic disease. These data are consistent with a role for CD8(+) T cells in atopic disease pathogenesis and may provide a basis for future T cell immunotherapy strategies.

van Baarle D, Kostense S, Hovenkamp E, Ogg G, Nanlohy N, Callan MF, Dukers NH, McMichael AJ, van Oers MH, Miedema F. 2002. Lack of Epstein-Barr virus- and HIV-specific CD27- CD8+ T cells is associated with progression to viral disease in HIV-infection. AIDS, 16 (15), pp. 2001-2011. | Show Abstract | Read more

OBJECTIVE: Despite readily detectable virus-specific CD8+ T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein-Barr virus (EBV)-specific CD8+ T cells was performed. DESIGN: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV-associated non-Hodgkin lymphoma's (NHL), both number and phenotype of virus-specific CD8+ T cells was studied longitudinally. METHODS: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells. RESULTS: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27- T cells, in contrast to rising numbers of EBV-specific CD27- CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS-related NHL had very low numbers of EBV-specific CD27- CD8+ T cells. Higher numbers of HIV-specific CD27- CD8+ T cells were associated with delayed disease progression. Virus-specific CD27- T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function. CONCLUSIONS: Taken together, our data indicate that virus-specific CD27- T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27- effector T cells may lead to progression of viral disease.

Dunbar PR, Ogg GS. 2002. Oligomeric MHC molecules and their homologues: state of the art. J Immunol Methods, 268 (1), pp. 3-7. | Show Abstract | Read more

This special issue of the Journal of Immunological Methods brings together articles from some of the leaders in labelling antigen-specific T and NKT cells, describing recent technical advances and their impact on the study of immunology. Although tetramers, or tetrameric MHC class I/peptide complexes, are the best known reagents in the field, various forms of oligomeric complexes are now being successfully used to detect antigen-specific T cells, including cytotoxic T lymphocytes, MHC class II-restricted CD4+ T cells, and glycolipid-specific T cells restricted by CD1 isoforms. The articles presented here detail the breadth of the oligomeric structures being used to probe T, NK and NKT cell function, and cover both the technical and practical aspects of their use, as well as the new biology revealed. In addition to providing a summary of the current state of the art, these contributions also provide clear pointers to strategies likely to succeed in the future. In this introductory chapter, we summarise the work presented in the other articles of this issue, and provide an overarching view of this rapidly evolving field. We also provide a summary of the MHC class I molecules successfully refolded to date, and provide references to other relevant sources of technical information.

Reignat S, Webster GJ, Brown D, Ogg GS, King A, Seneviratne SL, Dusheiko G, Williams R, Maini MK, Bertoletti A. 2002. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J Exp Med, 195 (9), pp. 1089-1101. | Show Abstract | Read more

Deletion, anergy, and a spectrum of functional impairments can affect virus-specific CD8 cells in chronic viral infections. Here we characterize a low frequency population of CD8 cells present in chronic hepatitis B virus (HBV) infection which survive in the face of a high quantity of viral antigen. Although they do not appear to exert immunological pressure in vivo, these CD8 cells are not classically "tolerant" since they proliferate, lyse, and produce antiviral cytokines in vitro. They are characterized by altered HLA/peptide tetramer reactivity, which is not explained by TCR down-regulation or reduced functional avidity and which can be reversed with repetitive stimulation. CD8 cells with altered tetramer binding appear to have a specificity restricted to envelope antigen and not to other HBV antigens, suggesting that mechanisms of CD8 cell dysfunction are differentially regulated according to the antigenic form and presentation of individual viral antigens.

Savage P, Cowburn P, Clayton A, Man S, McMichael A, Lemoine N, Epenetos A, Ogg G. 2002. Induction of viral and tumour specific CTL responses using antibody targeted HLA class I peptide complexes. Br J Cancer, 86 (8), pp. 1336-1342. | Show Abstract | Read more

The production of cytotoxic T cells with specificity for cancer cells is a rapidly evolving branch of cancer therapeutics. A variety of approaches aim to amplify anti-tumour cytotoxic T cell responses using purified peptides, tumour cell lysates or recombinant HLA/peptide complexes in differing antigen presenting systems. Using a two-step biotin-streptavidin antibody targeting system, recombinant HLA-class I/peptide complexes were attached to the surface of B cells via the anti-CD20 B9E9-scFvSA antibody-streptavidin fusion protein. Flow cytometry with a conformation dependant monoclonal antibody to HLA class I indicated that targeted HLA-class I/peptide complexes remain on the surface of B cells in culture for periods in excess of 72 h. PBMCs were stimulated in vitro for 8-14 days using the autologous B cells as antigen presenting cells. Following a single cycle of stimulation specific cytotoxic T cell responses to targeted HLA-A2 complexes containing the M1, BMLF1 and Melan A peptides could be demonstrated by tetramer staining and Cr release assays. With the HLA-A2/BMLF1 complex up to 2.99% of CD8+ve cells were tetramer positive producing 20% lysis (E : T 10 : 1) of CIR-A2 target cells in an in vitro cytotoxicity assay compared to baseline levels of 0.09% tetramer +ve and 2% lysis in the unstimulated population. PBMCs from a healthy donor treated with two cycles of stimulations with targeted HLA-A2/Melan A complexes, demonstrated expansion of the melanA tetramer +ve population from 0.03% to 1.4% producing 15% lysis of Melan A pulsed target cells. With further consideration to the key variables of HLA/peptide complex density, the ratio of stimulator to effector cells and optimum cytokine support, this system should offer an easy and effective method for the in vitro amplification of specific cytotoxic T cell responses and warrants development for the in vivo induction of cytotoxic T cell responses in cancer therapy.

Savage P, Cowburn P, Clayton A, Man S, Lawson T, Ogg G, Lemoine N, McMichael A, Epenetos A. 2002. Anti-viral cytotoxic T cells inhibit the growth of cancer cells with antibody targeted HLA class I/peptide complexes in SCID mice. Int J Cancer, 98 (4), pp. 561-566. | Show Abstract | Read more

A number of experimental antibody mediated cancer therapies aim to redirect cytotoxic T cells (CTLs) of non-tumour specificity to cancer cells. It has been previously demonstrated that cancer cells targeted with recombinant HLA-class I/viral peptide complexes via antibody delivery systems can be killed by virus specific CTLs. This novel therapeutic system has been developed with a simple pre-clinical model using the recombinant anti-CD20 B9E9 scFvSA fusion protein to target HLA-A2/peptide complexes to CD20 +ve Daudi lymphoma cells. In vitro data confirmed that, although binding of the B9E9 scFvSA fusion protein alone to Daudi cells had no effect on their growth, effective CTL mediated killing of Daudi cells could be achieved by targeting with B9E9 sfvScSA and recombinant HLA-A2/MI complexes at dilutions as low as 100 pg/ml. In contrast the free HLA-A2/MI complexes only significantly inhibited CTL activity at concentrations in excess of 100 ng/ml. The in vivo tumour protection assays in SCID mice demonstrated that only 1 of the 4 mice that received anti-HLA-A2/M1 CTLs and Daudi cells targeted with the B9E9 scFvSA fusion protein and HLA-A2/M1 complexes developed a tumour. In contrast in the control mice that received CTL and native Daudi cells all 4 developed tumours, as did all 4 that received targeted Daudi cells but no CTLs. Similar results were obtained in a parallel experiment using Daudi cells targeted with B9E9 scFvSA and HLA-A2/BMLF1 complexes and a CTL line to HLA-A2/BMLF1. The demonstration of in vivo activity for targeted HLA class I/peptide complexes combined with anti-viral T cells, supports the further clinical development of the system where it may be combined with autologous CTLs produced by vaccination or ex vivo expansion.

Appay V, Papagno L, Spina CA, Hansasuta P, King A, Jones L, Ogg GS, Little S, McMichael AJ, Richman DD, Rowland-Jones SL. 2002. Dynamics of T cell responses in HIV infection. J Immunol, 168 (7), pp. 3660-3666. | Show Abstract

Cytotoxic CD8(+) T cells play a major role in the immune response against viruses. However, the dynamics of CD8(+) T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8(+) T cells, in relation to Ag-specific CD4(+) T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8(+) T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.

Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, Ogg GS, King A 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.

Palmer RA, Ogg G, Allen J, Banerjee A, Ryatt KS, Ratnavel R, Wojnarowska F. 2001. Vancomycin-induced linear IgA disease with autoantibodies to BP180 and LAD285. Br J Dermatol, 145 (5), pp. 816-820. | Show Abstract | Read more

Linear IgA disease (LAD) is an acquired autoimmune subepidermal bullous disease characterized by the linear deposition of IgA at the basement membrane zone. A minority of cases are induced by drugs, of which the most frequently implicated is vancomycin. The target antigens in idiopathic LAD are heterogeneous, but have not previously been reported in vancomycin-induced LAD. We report three cases, and in two of these we investigated the target antigens. In both we identified IgA antibodies to LAD285 and IgA and IgG antibodies (dual response) to BP180.

Reignat S, Webster G, Brown D, Ogg G, Williams R, Dusheiko G, Maini M, Bertoletti A. 2001. HBV-specific CD8 cells show altered MHC/peptide binding in the presence of high levels of circulating viral antigen. HEPATOLOGY, 34 (4), pp. 609A-609A.

Wodarz D, Hall SE, Usuku K, Osame M, Ogg GS, McMichael AJ, Nowak MA, Bangham CR. 2001. Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1. Proc Biol Sci, 268 (1473), pp. 1215-1221. | Show Abstract | Read more

The correlation between virus load and specific cytotoxic T-lymphocyte (CTL) frequency during the chronic phase in human immunodeficiency virus type 1 (HIV-1) infection has been found to be negative in cross-sectional studies. We report here that, in infection with the related retrovirus human T-cell leukaemia virus type 1 (HTLV-1), the correlation is positive in asymptomatic carriers and zero in patients with the associated inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We demonstrate that the direction of the correlation may depend on the efficacy of the CTL response using mathematical models. We conclude that the CTL response is effective in asymptomatic carriers of HTLV-1, but ineffective in patients with HAM/TSP. Virus-mediated impairment of specific CTL production in HIV-1 infection can account for the negative correlation observed.

McDermott AB, Spiegel HM, Irsch J, Ogg GS, Nixon DF. 2001. A simple and rapid magnetic bead separation technique for the isolation of tetramer-positive virus-specific CD8 T cells. AIDS, 15 (6), pp. 810-812. | Read more

Boni C, Penna A, Ogg GS, Bertoletti A, Pilli M, Cavallo C, Cavalli A, Urbani S et al. 2001. Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy. Hepatology, 33 (4), pp. 963-971. | Show Abstract | Read more

The hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) response in patients with chronic HBV infection is generally weak or totally undetectable. This inability to mount protective CTL responses is believed to be a crucial determinant of viral persistence, and its correction represents an important objective of immune therapies for chronic hepatitis B. However, amplification of CTL responses in vivo may be ineffective if HBV-specific CD8 cells are either absent or nonresponsive to exogenous stimulation. In this study, we asked whether antiviral treatments able to inhibit viral replication and to reduce viral and antigen load can successfully reconstitute CTL responses creating the appropriate conditions for their therapeutic stimulation. For this purpose, the HBV-specific CTL response before and during lamivudine therapy was studied longitudinally in 6 HLA-A2-positive patients with HBeAg+ chronic hepatitis B. Both HBV-specific cytotoxic T cell activity measured by chromium release assay on peptide stimulation in vitro and CD8+ T cell frequency measured ex vivo by HLA-A2/peptide tetramer staining were significantly augmented by lamivudine therapy. This enhancement followed the reconstitution of CD4 reactivity and the decline of viral load induced by therapy. Our study shows that lamivudine treatment in chronic hepatitis B can restore CTL reactivity, making CTL susceptible to exogenous stimulation. This effect may enhance the probability that T cell-based immune therapies delivered after lamivudine treatment can successfully reconstitute a protective CTL response able to cure chronic HBV infection.

Klein MR, Smith SM, Hammond AS, Ogg GS, King AS, Vekemans J, Jaye A, Lukey PT, McAdam KP. 2001. HLA-B*35-restricted CD8 T cell epitopes in the antigen 85 complex of Mycobacterium tuberculosis. J Infect Dis, 183 (6), pp. 928-934. | Show Abstract | Read more

Few target epitopes have been described for human CD8 T lymphocytes in antigens of Mycobacterium tuberculosis. By use of a reverse immunogenetics approach, 23 motif-bearing peptides of the Ag85 complex were tested for binding to HLA-B*35, one of the common B-types in West Africa. Three 9-mer peptides bound with high affinity to HLA-B*3501 and displayed low dissociation rates of peptide-major histocompatibility complexes (MHCs). IC(50) and half-life values of peptide-MHC class I complexes were in the same range as reported earlier for other immunogenic peptides. Immune responses against peptide Ag85C (aa 204-212) WPTLIGLAM were characterized in detail. Peptide-stimulated effector cells were able to kill macrophages infected with M. tuberculosis or bacille Calmette-Guérin. Peptide-specific CD8 T cells could be visualized by using HLA-B*3501 tetramers and were shown to produce interferon-gamma and tumor necrosis factor-alpha. Together with other published epitopes, these peptides can be used to study more closely the role of CD8 T cells in mycobacterial infection and tuberculosis.

Webster GJM, Reignat S, Malacarne F, Brown D, Ogg G, Lascar M, Williams R, Dusheiko GM, Bertoletti A. 2001. Distinct functional patterns of HBV-specific CD8+cell response in hepatitis B virus infection GUT, 48 pp. A107-A107.

Kostense S, Ogg GS, Manting EH, Gillespie G, Joling J, Vandenberghe K, Veenhof EZ, van Baarle D, Jurriaans S, Klein MR, Miedema F. 2001. High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function. Eur J Immunol, 31 (3), pp. 677-686. | Show Abstract | Read more

To investigate the effect of HIV-specific CD8(+) T cells on viral plasma load and disease progression, we enumerated HLA-A2-, B8- and B57-restricted CD8(+) T cells directed against several HIV epitopes in a total of 54 patients by the use of tetrameric HLA-peptide complexes. In patients with high CD4(+) T cell numbers, HIV-specific tetramer(+) cells inversely correlated with viral load. Patients with CD4(+) T cell numbers below 400/microl blood, however, carried high viral load despite frequently having high tetramer(+) T cell numbers. This lack of correlation between viral load and tetramer(+) cells did not result from viral escape variants, as in only 4 of 13 patients, low frequencies of viruses with mutated epitopes were observed. In 15 patients we measured CD8(+) T cell antigen responsiveness to HIV peptide stimulation in vitro. FACS analyses showed differential IFN-gamma production of the tetramer(+) cells, and this proportion of IFN-gamma-producing tetramer(+) cells correlated with AIDS-free survival and with T cell maturation to the CD27(-) effector stage. These data show that most HIV-infected patients have sustained HIV-specific T cell expansions but many of these cells seem not to be functional, leaving the patient with high numbers of non-functional virus-specific CD8(+) T cells in the face of high viral burden.

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Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, Appay V, Rizzardi GP et al. 2001. Skewed maturation of memory HIV-specific CD8 T lymphocytes NATURE, 410 (6824), pp. 106-111. | Read more

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Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, Conlon C, Workman C, Shaunak S et al. 2001. Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses JOURNAL OF EXPERIMENTAL MEDICINE, 193 (3), pp. 375-385. | Show Abstract | Read more

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263-272, KR WIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, Conlon C, Workman C, Shaunak S et al. 2001. Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J Exp Med, 193 (3), pp. 375-386. | Show Abstract | Read more

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263-272, KRWIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

Komanduri KV, Donahoe SM, Moretto WJ, Schmidt DK, Gillespie G, Ogg GS, Roederer M, Nixon DF, McCune JM. 2001. Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects. Virology, 279 (2), pp. 459-470. | Show Abstract | Read more

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.

Goulder PJ, Tang Y, Brander C, Betts MR, Altfeld M, Annamalai K, Trocha A, He S et al. 2000. Functionally inert HIV-specific cytotoxic T lymphocytes do not play a major role in chronically infected adults and children. J Exp Med, 192 (12), pp. 1819-1832. | Show Abstract | Read more

The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

Smith SM, Brookes R, Klein MR, Malin AS, Lukey PT, King AS, Ogg GS, Hill AV, Dockrell HM. 2000. Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A. J Immunol, 165 (12), pp. 7088-7095. | Show Abstract

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.

Roy MJ, Wu MS, Barr LJ, Fuller JT, Tussey LG, Speller S, Culp J, Burkholder JK et al. 2000. Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine. Vaccine, 19 (7-8), pp. 764-778. | Show Abstract | Read more

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-naïve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.

Webster GJ, Reignat S, Maini MK, Whalley SA, Ogg GS, King A, Brown D, Amlot PL et al. 2000. Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology, 32 (5), pp. 1117-1124. | Show Abstract | Read more

After hepatitis B virus (HBV) infection, liver injury and viral control have been thought to result from lysis of infected hepatocytes by virus-specific cytotoxic T cells. Patients are usually studied only after developing significant liver injury, and so the viral and immune events during the incubation phase of disease have not been defined. During a single-source outbreak of HBV infection, we identified patients before the onset of symptomatic hepatitis. The dynamics of HBV replication, liver injury, and HBV-specific CD8+ and CD4+ cell responses were investigated from incubation to recovery. Although a rise in alanine transaminase (ALT) levels was present at the time of the initial fall in HBV-DNA levels, maximal reduction in virus level occurred before significant liver injury. Direct ex vivo quantification of HBV-specific CD4+ and CD8+ cells, by using human leukocyte antigen (HLA) class I tetramers and intracellular cytokine staining, showed that adaptive immune mechanisms are present during the incubation phase, at least 4 weeks before symptoms. The results suggest that the pattern of reduction in HBV replication is not directly proportional to tissue injury during acute hepatitis B in humans. Furthermore, because virus-specific immune responses and significant reductions in viral replication are seen during the incubation phase, it is likely that the immune events central to viral control occur before symptomatic disease.

Maini MK, Reignat S, Boni C, Ogg GS, King AS, Malacarne F, Webster GJ, Bertoletti A. 2000. T cell receptor usage of virus-specific CD8 cells and recognition of viral mutations during acute and persistent hepatitis B virus infection. Eur J Immunol, 30 (11), pp. 3067-3078. | Show Abstract | Read more

T cells specific for a single viral epitope, but using different T cell receptors, should have flexibility in their epitope recognition to protect the infected host against the emergence of viral escape mutants. Therefore, polyclonality of the hepatitis B virus (HBV)-specific cytotoxic T lymphocyte response has been hypothesized to be a major determinant in the control of infection. We analyzed the Vbeta chain composition of the core 18-27-specific CD8 cells in acute and persistently HBV-infected patients using HLA-A2 tetrameric complexes and a panel of Vbeta antibodies. Different T cell receptors were utilized by core 18-27-specific CD8 cells both in patients with acute and chronic infection. The functional ability of these epitope-specific T cells to respond to potential viral mutations was then tested. The polyclonal HBV-specific CD8 response present in patients with acute hepatitis displayed a limited efficiency to recognize mutations introduced within the epitope. The ability of core 18-27-specific CD8 to tolerate epitope mutations was found only during persistent HBV infection. The data suggest that although a clonally heterogeneous CD8 response can be largely inhibited by the occurrence of single epitope mutations in primary HBV infection, preferential selection of T cells able to counteract the emergence of viral mutations can occur during persistent infection.

Tomiyama H, Oka S, Ogg GS, Ida S, McMichael AJ, Takiguchi M. 2000. Expansion of HIV-1-specific CD28- CD45RA- CD8+ T cells in chronically HIV-1-infected individuals. AIDS, 14 (13), pp. 2049-2051. | Read more

Jin X, Ogg G, Bonhoeffer S, Safrit J, Vesanen M, Bauer D, Chen D, Cao Y et al. 2000. An antigenic threshold for maintaining human immunodeficiency virus type 1-specific cytotoxic T lymphocytes. Mol Med, 6 (9), pp. 803-809. | Show Abstract

BACKGROUND: Using the lymphocytic choriomeningitis virus (LCMV) model in mice, a number of studies show that memory cytotoxic T-lymphocyte (CTL) responses are maintained in the presence of continuous antigenic stimulation. Yet, other groups found that memory CTL specific for LCMV could last for a lifetime in mice without viral antigens. Thus, the extent to which an antigen is required for the maintenance of virus-specific CTL remains controversial. In humans, very few studies have been conducted to investigate the relationship between the quantity of antigen and the magnitude of CTL responses. MATERIALS AND METHODS: We quantified CTL precursors (CTLp) using a limiting-dilution analysis (LDA) and CTL effectors (CTLe) using a new Major Histocompatibility Complex (MHC) class I tetramer technology in six long-term nonprogressors (LTNPs) with human immunodeficiency virus type-1 (HIV-1) infection, as well as in eight patients whose viral loads were well suppressed by antiretroviral therapy. The viremia levels in these patients were measured using an reverse transcription polymerase chain reaction (RT-PCR) assay. The proviral DNA load in peripheral blood mononuclear cell (PBMC) was also measured by PCR in four LTNPs. RESULTS: The LTNPs had high levels of HIV-1-specific memory CTLp and CTLe, while maintaining a low plasma viral load. Despite also having low viral loads, patients whose plasma viremia was well-suppressed by effective therapy had low levels of CTLe. CONCLUSIONS: Our findings suggest that a complex, rather than a monotonic, relationship exists between CTL levels and HIV-1 viremia, including what appears to be an antigenic threshold for the maintenance of CTL at a measurable level. Under conditions of "antigen excess,", CTLe levels correlate inversely with viral load. On the other hand, under conditions that are "antigen limited," the correlation appears to be direct.

McMichael AJ, Callan M, Appay V, Hanke T, Ogg G, Rowland-Jones S. 2000. The dynamics of the cellular immune response to HIV infection: implications for vaccination. Philos Trans R Soc Lond B Biol Sci, 355 (1400), pp. 1007-1011. | Show Abstract | Read more

Recent advances in measuring T-cell responses to viruses have led to new insights into how these T cells respond. In the acute infection there are massive CD8+ T-cell responses to both Epstein-Barr virus (EBV) and to human immunodeficiency virus (HIV). Many of these T cells are effector cells and only a minority appear to be capable of maintaining immunological memory. In persistent virus infections, high levels of antigen-specific effector cells persist. If virus does not persist, the effectors fade in number but memory is maintained and is primed to react rapidly to a new challenge. A vaccine that stimulates only T-cell responses may protect when these memory cells respond rapidly enough to generate high numbers of effectors before the infecting virus becomes established.

Barnardo MC, Harmer AW, Shaw OJ, Ogg GS, Bunce M, Vaughan RW, Morris PJ, Welsh KI. 2000. Detection of HLA-specific IGG antibodies using single recombinant HLA alleles: the MonoLISA assay. Transplantation, 70 (3), pp. 531-536. | Show Abstract | Read more

BACKGROUND: Because of the presence of confounding antigens, the assignment of HLA antibody specificity is difficult in highly sensitized patients, and the definition of an acceptable HLA mismatch requires a significant workload per patient. We describe a new ELISA method, monoLISA, for detection of immunoglobulin (Ig)G HLA antibody using single recombinant HLA class I monomers bound to microtiter plates. METHODS: HLA-A2 and -B8 monomers were synthesized and used as screening targets for 85 sera from renal patients. The sera contained various IgG and IgM HLA-specific antibodies, including anti-A2 and anti-B8,defined in a conventional complement-dependent cytotoxicity test (CDC). Investigations were performed to determine possible effects on antibody binding of differential monomer peptide presentation as well as lack of glycosylation. RESULTS: A good correlation was found between CDC-defined specificities and the reactivity observed with HLA monomers. MonoLISA attained means of 100% sensitivity and 92.5% specificity compared with CDC. Neither the presence of different peptides, nor the absence of glycosylation of the monomer affected the ability of monoLISA to detect antibody. CONCLUSION: This study demonstrates that the mono-LISA method for HLA antibody detection is valid. Because this has the potential to reduce the work involved in screening sensitized patients awaiting transplantation for HLA antibodies, resources aimed at increasing the number of constructed monomers would be well targeted.

Shacklett BL, Beadle TJ, Pacheco PA, Grendell JH, Haslett PA, King AS, Ogg GS, Basuk PM, Nixon DF. 2000. Isolation of cytomegalovirus-specific cytotoxic T-lymphocytes from gut-associated lymphoid tissue (GALT) of HIV type 1-infected subjects. AIDS Res Hum Retroviruses, 16 (12), pp. 1157-1162. | Show Abstract | Read more

Cytomegalovirus (CMV) can be an important opportunistic infection in HIV-1-infected patients, particularly when the CD4+ T-cell count drops below 50 lymphocytes/mm3. CMV-associated disease, including retinitis, pneumonitis, gastroenteritis, and encephalitis, is estimated to affect up to 40% of AIDS patients. We have studied the cellular immune response to CMV in gut-associated lymphoid tissue (GALT) of HIV-1-infected patients. Two patients with chronic diarrhea of unknown etiology were examined by flexible sigmoidoscopy and upper endoscopy. Biopsy specimens were obtained from lymphoid-associated tissue sites in rectum and duodenum. Both patients were seropositive for CMV IgG, but had not been treated with ganciclovir, and neither had clinical signs of CMV disease. Mononuclear cell cultures were established from GALT and blood and assayed for the presence of CMV-specific CD8+ T cells. CD8+ T-cell phenotype and function were assessed by MHC Class I tetramer staining, using an HLA-A*0201 tetramer complex specific for peptide 495-503 (NLVPMVATV) of CMV lower matrix protein pp65, and by a standard 51Cr release assay. CMV pp65-specific cytotoxic lymphocytes (CTL) were detected in GALT and blood MNC from both patients. These results demonstrate that HIV-1-infected subjects seropositive for CMV, but without active CMV gastrointestinal disease, harbor CMV-specific CTL in intestinal lymphoid tissue. This is the first report of isolation of CMV-specific CTL in GALT and will lead to greater understanding of the pathogenesis of CMV disease in human mucosal tissue.

Appay V, Nixon DF, Donahoe SM, Gillespie GM, Dong T, King A, Ogg GS, Spiegel HM et al. 2000. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J Exp Med, 192 (1), pp. 63-75. | Show Abstract | Read more

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Ogg GS. 2000. Detection of antigen-specific cytotoxic T lymphocytes: significance for investigative dermatology. Clin Exp Dermatol, 25 (4), pp. 312-316. | Show Abstract | Read more

Novel recombinant tetrameric complexes of HLA class I molecules allow the direct visualization of antigen-specific CD8+ T cells using flow cytometry. By facilitating the quantification, isolation and phenotypic analysis of CD8+ T cells, the use of HLA tetramers has extended our understanding of the role of cellular immunity in various disease settings. Recently the technique has also been applied to the study of cutaneous disease and provides insights into mechanisms of dermatopathology.

Goulder PJ, Brander C, Annamalai K, Mngqundaniso N, Govender U, Tang Y, He S, Hartman KE et al. 2000. Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children. J Virol, 74 (12), pp. 5679-5690. | Show Abstract | Read more

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17(Gag) and p24(Gag) sequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17(Gag) peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24(Gag) peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%; P < 0.005). Within this 20-mer p24(Gag), an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.

Shacklett BL, Beadle TJ, Pacheco PA, Grendell JH, Haslett PA, King AS, Ogg GS, Basuk PM, Nixon DF. 2000. Characterization of HIV-1-specific cytotoxic T lymphocytes expressing the mucosal lymphocyte integrin CD103 in rectal and duodenal lymphoid tissue of HIV-1-infected subjects. Virology, 270 (2), pp. 317-327. | Show Abstract | Read more

Acute HIV-1 infection depletes CD4(+) T cells in gut-associated lymphoid tissue (GALT). The failure of containment of local viral replication, and consequent CD4(+) T cell depletion, might be due to delayed mobilization of effector CD8(+) T cells or absence of functioning HIV-1-specific CD8(+) T cell effectors within GALT. No studies have addressed human intestinal HIV-1-specific CD8(+) T cell functions. We sought to determine whether functional HIV-1-specific CTL were present in GALT and whether the repertoire differed from HIV-1-specific CTL isolated from peripheral blood mononuclear cells. From three HIV-1-infected subjects, we isolated HIV-1-specific CD8(+) T cells expressing the mucosal lymphocyte integrin CD103 from GALT. These antigen-specific effector cells could be expanded in vitro and lysed target cells in an MHC class I-restricted manner. HIV-1-specific CTL could be isolated from both duodenal and rectal GALT sites, indicating that CD8(+) effectors were widespread through GALT tissue. The breadth and antigenic specificities of GALT CTL appeared to differ from those in peripheral blood in some cases. In summary, we found HIV-1-specific CD8(+) effector T cells in GALT, despite HIV-1-induced CD4(+) T cell lymphopenia. This suggests that HIV-1-specific CTL in gut tissue can be maintained with limited CD4(+) T cell help.

Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, King AS, Herberg J et al. 2000. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med, 191 (8), pp. 1269-1280. | Show Abstract | Read more

Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.

McMichael AJ, Ogg G, Wilson J, Callan M, Hambleton S, Appay V, Kelleher T, Rowland-Jones S. 2000. Memory CD8+ T cells in HIV infection. Philos Trans R Soc Lond B Biol Sci, 355 (1395), pp. 363-367. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent HIV infection in humans. The kinetics and general features of the CTL response are similar to those found during other persisting virus infections in humans. During chronic infection there are commonly between 0.1 and 1.0% of all CD8+ T cells in the blood that are specific for immunodominant virus epitopes, as measured by HLA class I peptide tetramers. These figures are greatly in excess of the numbers found by limiting dilution assays; the discrepancy may arise because in the latter assay, CTLs have to divide many times to be detected and many of the HIV-specific CD8+ T cells circulating in infected persons may be incapable of further division. Many tetramer-positive T cells make interferon-gamma, beta-chemokines and perforin, so are probably functional. It is not known how fast these T cells turn over, but in the absence of antigen they decay in number. Impairment of CTL replacement, because CD4+ T helper cells are depleted by HIV infection, may play a major role in the development of AIDS.

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.

Wilson JD, Ogg GS, Allen RL, Davis C, Shaunak S, Downie J, Dyer W, Workman C, Sullivan S, McMichael AJ, Rowland-Jones SL. 2000. Direct visualization of HIV-1-specific cytotoxic T lymphocytes during primary infection. AIDS, 14 (3), pp. 225-233. | Show Abstract | Read more

OBJECTIVE: HIV-specific cytotoxic T lymphocytes (CTL) are believed to play an important role in containing viral replication throughout HIV-1 infection. Previous studies have attempted to quantify the HIV-1-specific CTL precursor frequency during primary HIV infection by using limiting dilution analysis, which almost certainly underestimates the true CTL frequency. Here we use a relatively new technique to quantify HIV-specific CD8 T cells in primary HIV infection. METHODS: We have used soluble tetrameric complexes of HLA class I molecules complexed with HIV epitope peptides to study the dynamics and frequency of HIV-specific CD8 T cells in relation to plasma viral load in early HIV infection, in three patients with a highly focused HIV-specific CTL response. RESULTS: We show that the frequencies of HIV-1-specific CD8 T cells in acute infection are significantly higher than previously documented and can be demonstrated well before full seroconversion. These studies also confirm the immunodominance of the B27-restricted response in HIV infection and demonstrate a close temporal relationship between the numbers of circulating HIV-specific CD8 T cells and viral load. CONCLUSIONS: These findings strongly suggest that HIV-1-specific CD8 T cells are responding directly to the level of viral replication in early HIV infection and are a major factor in its control. In addition, the data indicate that immunodominance for CD8 T-cell responses is established in the acute phase of HIV infection.

Larrubia JR, Herberg JA, Reignat S, Ogg GS, Webster G, Williams R, Maini MK, Bertoletti A. 2000. Chemokine receptor expression and cytokine production of HBV-specific CD8 cells JOURNAL OF HEPATOLOGY, 32 pp. 90-90. | Read more

Webster GJM, Reignat S, Maini MK, Brown D, Whalley SA, Ogg GS, Williams R, Vergani D, Dusheiko GM, Bertoletti A. 2000. Dynamics of HBV-specific CD4 and CD8 response during early phase of acute hepatitis B JOURNAL OF HEPATOLOGY, 32 pp. 90-90. | Read more

Maini MK, Boni C, Lee K, Larrubia JR, Reignat S, Ogg GS, Herberg J, Gilson R et al. 2000. The role of virus-specific CD8+cells in liver damage and viral control during persistent hepatitis B virus infection JOURNAL OF HEPATOLOGY, 32 pp. 46-46. | Read more

Spiegel HM, Ogg GS, DeFalcon E, Sheehy ME, Monard S, Haslett PA, Gillespie G, Donahoe SM et al. 2000. Human immunodeficiency virus type 1- and cytomegalovirus-specific cytotoxic T lymphocytes can persist at high frequency for prolonged periods in the absence of circulating peripheral CD4(+) T cells. J Virol, 74 (2), pp. 1018-1022. | Show Abstract | Read more

CD4(+) T cells are thought to be critical in the maintenance of virus-specific CD8(+) cytotoxic T-cell (CTL) responses. In human immunodeficiency virus type 1 (HIV-1) infection, a selective decline in HIV-1-specific CTL as the CD4(+) T-cell count decreases has been reported. Using HLA-peptide tetrameric complexes, we show the presence at high frequency of HIV-1- and cytomegalovirus-specific CD8(+) T cells when the peripheral CD4(+) T-cell count was low or zero in three HIV-1-infected patients. No direct virus-specific CD8(+)-mediated effector activity was seen in these subjects, suggesting antigen unresponsiveness, although tetramer-sorted cells could be expanded in vitro in the presence of interleukin-2 into responsive effector cells. Thus, virus-specific CD8(+) T cells can be maintained in the peripheral circulation at high frequency in the absence of circulating peripheral CD4(+) T cells, but these cells may lack direct effector activity. Strategies designed to overcome this antigen unresponsiveness may be of value in therapies for the treatment of AIDS.

Maini MK, Boni C, Ogg GS, King AS, Reignat S, Lee CK, Larrubia JR, Webster GJ et al. 1999. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology, 117 (6), pp. 1386-1396. | Show Abstract | Read more

BACKGROUND & AIMS: Cytotoxic T cells have been suggested to be responsible for lysis of hepatitis B virus (HBV)-infected hepatocytes and control of virus infection. The frequency, kinetics, phenotype, and capacity for clonal expansion of circulating HBV-specific CD8 cells were analyzed directly in patients with acute HBV infection to clarify their pathogenetic role. METHODS: Three HLA-A2 peptide tetramers able to visualize HBV core, envelope, and polymerase epitope-specific cytotoxic T lymphocytes were synthesized and used for flow cytometric analysis of antigen-specific populations. RESULTS: Tetramer-positive cells specific for the core 18-27 epitope were found at a higher frequency than those specific for polymerase 575-583 and envelope 335-343 epitopes in most patients with acute HBV. The number of HBV-specific CD8 cells was highest during the clinically acute stage of infection and decreased after recovery. These cells expressed an activated phenotype and had an impaired capacity to expand in vitro and to display cytolytic activity in response to peptide stimulation. Recovery of these functions was observed when the frequency of specific CD8 cells decreased, coincident with a progressive decrease in their expression of activation markers. CONCLUSIONS: This study provides the first ex vivo evidence that the highest frequency of circulating HBV-specific CD8 cells coincides with the clinically acute phase of hepatitis B. These cells exhibit an activated phenotype with limited further proliferative capacity that is restored during recovery.

Ogg GS, Kostense S, Klein MR, Jurriaans S, Hamann D, McMichael AJ, Miedema F. 1999. Longitudinal phenotypic analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes: correlation with disease progression. J Virol, 73 (11), pp. 9153-9160. | Show Abstract

Few studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8(+) T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17(Gag/Pol) tetramer-binding cells and slower disease progression (P < 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27(+) CD45RO(+) cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27(+) CD45RO(+) subset.

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.

Webster GJ, Whalley SA, Maini M, Brown D, Ogg G, Vergani D, Williams R, Dusheiko GM, Bertoletti A. 1999. Hepatocyte lysis by HBV-specific CD8 cells may not be the principal mechanism of viral control in acute hepatitis B. HEPATOLOGY, 30 (4), pp. 440A-440A.

Ogg GS, King AS, Dunbar PR, McMichael AJ. 1999. Isolation of HIV-1-specific cytotoxic T lymphocytes using human leukocyte antigen-coated beads. AIDS, 13 (14), pp. 1991-1993. | Read more

Kalams SA, Goulder PJ, Shea AK, Jones NG, Trocha AK, Ogg GS, Walker BD. 1999. Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy. J Virol, 73 (8), pp. 6721-6728. | Show Abstract

Therapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38(+) activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.

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.

Larsson M, Jin X, Ramratnam B, Ogg GS, Engelmayer J, Demoitie MA, McMichael AJ, Cox WI, Steinman RM, Nixon D, Bhardwaj N. 1999. A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS, 13 (7), pp. 767-777. | Show Abstract | Read more

OBJECTIVES: HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. RESULTS: In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. CONCLUSIONS: Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.

Maini MK, Webster GJM, Boni C, Ogg GS, Lee CK, Brown D, Reignat S, Dusheiko GM et al. 1999. Kinetics of the HBV-specific CTL response GUT, 44 pp. A52-A52.

Tan R, Xu X, Ogg GS, Hansasuta P, Dong T, Rostron T, Luzzi G, Conlon CP, Screaton GR, McMichael AJ, Rowland-Jones S. 1999. Rapid death of adoptively transferred T cells in acquired immunodeficiency syndrome. Blood, 93 (5), pp. 1506-1510. | Show Abstract

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) probably play the major role in controlling HIV replication. However, the value of adoptive transfer of HIV-specific CTL expanded in vitro to HIV+ patients has been limited: this contrasts with the success of CTL therapy in treating or preventing Epstein-Barr virus and cytomegalovirus disease after bone marrow transplantation (BMT). We investigated the fate of expanded HIV-specific CTL clones in vivo following adoptive transfer to a patient with acquired immunodeficiency syndrome (AIDS). Two autologous CTL clones specific for HIV Gag and Pol were expanded to large numbers (>10(9)) in vitro and infused into an HIV-infected patient whose viral load was rising despite antiretroviral therapy. The fate of one clone was monitored by staining peripheral blood mononuclear cells (PBMCs) with T-cell receptor-specific tetrameric major histocompatibility complex (MHC)-peptide complexes. Although the CTL transfer was well tolerated, there were no significant changes in CD4 and CD8 lymphocyte counts and virus load. By tracking an infused clone using soluble MHC-peptide complexes, we show that cells bearing the Gag-specific T-cell receptors were rapidly eliminated within hours of infusion through apoptosis. Thus, the failure of adoptively transferred HIV-specific CTL to reduce virus load in AIDS may be due to rapid apoptosis of the infused cells, triggered by a number of potential mechanisms. Further trials of adoptive transfer of CTL should take into account the susceptibility of infused cells to in vivo apoptosis.

Agrawal S, Marquet J, Freeman GJ, Tawab A, Bouteiller PL, Roth P, Bolton W, Ogg G, Boumsell L, Bensussan A. 1999. Cutting edge: MHC class I triggering by a novel cell surface ligand costimulates proliferation of activated human T cells. J Immunol, 162 (3), pp. 1223-1226. | Show Abstract

BY55 is a human cell surface molecule whose expression is restricted to NK cells, a subset of circulating CD8+ T lymphocytes, and all intestinal intraepithelial T lymphocytes. Here, we report that BY55 is a novel NK receptor showing broad specificity for both classical and nonclassical MHC class I molecules, and that optimal binding requires a prior aggregation of MHC class I complexes. Using BY55 transfectants, we have identified functional consequences of MHC class I/ligand interactions for the class I-bearing cell. The triggering of MHC class I molecules on human T cell clones by BY55 delivered a potent proliferative signal in the presence of soluble CD3 mAb. The costimulatory signal provided by MHC class I ligation was only seen in activated, and not resting, peripheral blood T cells. This observation represents an additional and/or alternative pathway to CD28 costimulation and may be of particular relevance in memory T cells lacking CD28, such as intestinal intraepithelial T lymphocytes, which are CD28- but BY55+.

Dorrell L, Dong T, Ogg GS, Lister S, McAdam S, Rostron T, Conlon C, McMichael AJ, Rowland-Jones SL. 1999. Distinct recognition of non-clade B human immunodeficiency virus type 1 epitopes by cytotoxic T lymphocytes generated from donors infected in Africa. J Virol, 73 (2), pp. 1708-1714. | Show Abstract

We present detailed studies of human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses to clade A or C HIV type 1 in three donors infected in East Africa. We define several novel non-clade B CTL epitopes, including some restricted by HLA alleles common in Africans. Although cross-clade CTL recognition of these epitopes does occur, recognition can also be highly clade specific.

Ogg GS, Jin X, Bonhoeffer S, Moss P, Nowak MA, Monard S, Segal JP, Cao Y et al. 1999. Decay kinetics of human immunodeficiency virus-specific effector cytotoxic T lymphocytes after combination antiretroviral therapy. J Virol, 73 (1), pp. 797-800. | Show Abstract

Little is known of the changes in human immunodeficiency virus type 1 (HIV-1)-specific effector cytotoxic T lymphocytes (CTL) after potent antiretroviral therapy. Using HLA/peptide tetrameric complexes, we show that after starting treatment, there are early rapid fluctuations in the HIV-1-specific CTL response which last 1 to 2 weeks. These fluctuations are followed by an exponential decay (median half-life, 45 days) of HIV-1-specific CTL which continues while viremia remains undetectable. These data have implications for the immunological control of drug-resistant virus.

Dyer WB, Ogg GS, Demoitie MA, Jin X, Geczy AF, Rowland-Jones SL, McMichael AJ, Nixon DF, Sullivan JS. 1999. Strong human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte activity in Sydney Blood Bank Cohort patients infected with nef-defective HIV type 1. J Virol, 73 (1), pp. 436-443. | Show Abstract

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with natural nef and nef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.

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.

Maini MK, Ogg GS, Boni C, Pilli M, Ferrari C, McMichael AJ, Williams R, Vergani D, Bertoletti A. 1998. Direct visualisation of hepatitis B virus (HBV)-specific cytotoxic T cells with an human leucocyte antigen (HLA) class I peptide tetrameric complex allows a new insight into their actual frequency. HEPATOLOGY, 28 (4), pp. 485A-485A.

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.

Ogg GS, Dong T, Hansasuta P, Dorrell L, Clarke J, Coker R, Luzzi G, Conlon C, McMichael AP, Rowland-Jones S. 1998. Four novel cytotoxic T-lymphocyte epitopes in the highly conserved major homology region of HIV-1 Gag, restricted through B*4402, B*1801, A*2601, B*70 (B*1509) AIDS, 12 (12), pp. 1561-1563. | Read more

Wilson JD, Ogg GS, Allen RL, Goulder PJ, Kelleher A, Sewell AK, O'Callaghan CA, Rowland-Jones SL, Callan MF, McMichael AJ. 1998. Oligoclonal expansions of CD8(+) T cells in chronic HIV infection are antigen specific. J Exp Med, 188 (4), pp. 785-790. | Show Abstract | Read more

Acute HIV infection is associated with a vigorous immune response characterized by the proliferation of selected T cell receptor V beta (BV)-expressing CD8(+) T cells. These 'expansions', which are commonly detected in the peripheral blood, can persist during chronic HIV infection and may result in the dominance of particular clones. Such clonal populations are most consistent with antigen-driven expansions of CD8(+) T cells. However, due to the difficulties in studying antigen-specific T cells in vivo, it has been hard to prove that oligoclonal BV expansions are actually HIV specific. The use of tetrameric major histocompatibility complex-peptide complexes has recently enabled direct visualization of antigen-specific T cells ex vivo but has not provided information on their clonal composition. We have now made use of these tetrameric complexes in conjunction with anti-BV chain-specific monoclonal antibodies and analysis of cytotoxic T lymphocyte lines/clones to show that chronically clonally expanded CD8(+) T cells are HIV specific in vivo.

Ogg GS, McMichael AJ. 1998. HLA-peptide tetrameric complexes. Curr Opin Immunol, 10 (4), pp. 393-396. | Show Abstract | Read more

HLA-peptide tetrameric complexes allow the direct ex vivo visualisation of antigen-specific CD8+ T cells and natural killer cells by flow cytometry. Quantitation, phenotypic analysis and isolation of tetramer-binding cells has considerably extended our understanding of cytotoxic T lymphocyte and natural killer cell function.

Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, O'Callaghan CA, Steven N, McMichael AJ, Rickinson AB. 1998. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo. J Exp Med, 187 (9), pp. 1395-1402. | Show Abstract | Read more

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex-peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8+ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8+ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.

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 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.

Braud VM, Allan DS, O'Callaghan CA, Söderström K, D'Andrea A, Ogg GS, Lazetic S, Young NT et al. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 391 (6669), pp. 795-799. | Show Abstract | Read more

The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.

Hanke T, Blanchard TJ, Schneider J, Ogg GS, Tan R, Becker M, Gilbert SC, Hill AV, Smith GL, McMichael A. 1998. Immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus Ankara-based multi-CTL epitope vaccine for human immunodeficiency virus type 1 in mice. J Gen Virol, 79 ( Pt 1) (1), pp. 83-90. | Show Abstract | Read more

A vaccine against human immunodeficiency virus (HIV) is still awaited. Although the correlates of protection remain elusive, it is likely that CD8+ T cells play an important role in the control of this infection. To firmly establish the importance of these cells in protective immunity, a means of efficient elicitation of CD8+ T cell responses in the absence of antibody is needed and, when available, might represent a crucial step towards a protective vaccine. Here, a novel vaccine candidate was constructed as a multi-cytotoxic T lymphocyte (CTL) epitope gene delivered and expressed using modified vaccinia virus Ankara (MVA). The immunogen consists of 20 human, one murine and three rhesus macaque epitopes. The non-human epitopes were included so that the vaccine can be tested for immunogenicity and optimal vaccination doses, routes and regimes in experimental animals. Mice were immunized intravenously (i.v.) or intramuscularly (i.m.) using a single dose of 10(6) p.f.u. of the recombinant MVA and the induction of CTL was assessed. It was demonstrated that both administration routes induced specific CTL responses and that the i.v. route was moderately more immunogenic than the i.m. route. The frequencies of ex vivo splenocytes producing interferon-y upon MHC class I-restricted peptide stimulation were determined using an ELISPOT assay. Also, the correct processing and presentation of some HLA-restricted epitopes in human cells was confirmed.

Lalvani A, Dong T, Ogg G, Patham AA, Newell H, Hill AV, McMichael AJ, Rowland-Jones S. 1997. Optimization of a peptide-based protocol employing IL-7 for in vitro restimulation of human cytotoxic T lymphocyte precursors. J Immunol Methods, 210 (1), pp. 65-77. | Show Abstract | Read more

A variety of different methods for the in vitro restimulation of human cytotoxic T lymphocyte (CTL) precursors (CTLp) are in use. Our aim was to enhance the detection of circulating human CTLp in peripheral blood. We have developed a standardized and highly efficient method for restimulating CTLp. Synthetic peptides were used to restimulate cognate CTLp from peripheral blood mononuclear cells (PBMC), and effector CTL capable of lysing peptide-pulsed and virus infected targets were generated. The effects of several parameters on CTL specific for influenza A, EBV and HIV-1 were evaluated, and the optimum peptide concentration for CTL generation was established. Supplementation of initial cultures with IL-7 greatly enhanced peptide-specific lytic activity for all peptides tested and the dose-response relationship for IL-7 was delineated. A novel technique using peptide-MHC class I molecule tetramers to stain T cells bearing cognate T cell receptors permitted enumeration of antigen-specific CD8 + CTL during in vitro restimulation; IL-7 supplementation selectively expanded the population of peptide-specific CD8 + CTL. Importantly, this protocol, whilst enhancing the restimulation and lytic activity of secondary CTL, does not induce primary CTL in vitro. The improved efficiency with which CTL are generated in this system substantially enhances the sensitivity of CTL culture and the 51Cr release assay to detect low levels of CTL activity.

Reich Z, Altman JD, Boniface JJ, Lyons DS, Kozono H, Ogg G, Morgan C, Davis MM. 1997. Stability of empty and peptide-loaded class II major histocompatibility complex molecules at neutral and endosomal pH: comparison to class I proteins. Proc Natl Acad Sci U S A, 94 (6), pp. 2495-2500. | Show Abstract | Read more

The structure and thermal stability of empty and peptide-filled forms of the murine class II major histocompatibility complex (MHC) molecule I-E(k) were studied at neutral and mildly acidic pH. The two forms have distinct circular dichroic spectra, suggesting that a conformational change may accompany peptide binding. Thermal stability profiles indicate that binding of peptide significantly increases the thermal stability of the empty heterodimers at both neutral and mildly acidic pH. Free energies calculated from these data provide a direct measure of this stabilization and show that the empty form of I-E(k) is significantly more stable than that of class I MHC proteins. Furthermore, for the two MHC class II proteins that were analyzed (I-E(k) and I-A(d)), thermal stability was not significantly altered by acidification. In contrast, of four class I MHC molecules studied, three have shown a significant loss in complex stability at low pH. The marked stability exhibited by their empty form, as well as their resistance to low pH, as observed in this study, correlate well with the ability of class II MHC molecules to traverse and bind peptides in acidic endosomal vesicles.

Goulder PJ, Phillips RE, Colbert RA, McAdam S, Ogg G, Nowak MA, Giangrande P, Luzzi G et al. 1997. Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat Med, 3 (2), pp. 212-217. | Show Abstract | Read more

The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial. Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops. It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape. The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection. We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope. In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability. CTL escape may play an important role in the pathogenesis of HIV infection.

Klenerman P, Phillips RE, Rinaldo CR, Wahl LM, Ogg G, May RM, McMichael AJ, Nowak MA. 1996. Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection. Proc Natl Acad Sci U S A, 93 (26), pp. 15323-15328. | Show Abstract | Read more

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells-at plausible peripheral blood mononuclear cell-to-target ratios-with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per microliter, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

McClure MO, Goulder PJ, Ogg G, McMichael AJ, Weber JN. 1996. HIV clearance in infants. Lancet, 347 (9008), pp. 1122. | Read more

Nixon DF, Broliden K, Ogg G, Broliden PA. 1992. Cellular and humoral antigenic epitopes in HIV and SIV. Immunology, 76 (4), pp. 515-534.

Bodmer H, Ogg G, Gotch F, McMichael A. 1989. Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes. Nature, 342 (6248), pp. 443-446. | Show Abstract | Read more

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.

Salimi M, Xue L, Jolin H, Hardman C, Cousins DJ, McKenzie AN, Ogg GS. 2016. Group 2 Innate Lymphoid Cells Express Functional NKp30 Receptor Inducing Type 2 Cytokine Production. J Immunol, 196 (1), pp. 45-54. | Show Abstract | Read more

Group 2 innate lymphoid cells (ILC2) are important in effector functions for eliciting allergic inflammation, parasite defense, epithelial repair, and lipid homeostasis. ILC2 lack rearranged Ag-specific receptors, and although many soluble factors such as cytokines and lipid mediators can influence ILC2, direct interaction of these cells with the microenvironment and other cells has been less explored. Natural cytotoxicity receptors are expressed by subsets of group 1 ILC and group 3 ILC and thought to be important for their effector function, but they have not been shown to be expressed by ILC2. Therefore, we sought to investigate the expression and functional properties of the natural cytotoxicity receptor NKp30 on human ILC2. A subset of ex vivo and cultured ILC2 express NKp30 that upon interaction with its cognate activatory ligand B7-H6 induces rapid production of type 2 cytokines. This interaction can be blocked by NKp30 blocking Ab and an inhibitory ligand, galectin-3. Higher expression of B7-H6 was observed in lesional skin biopsies of patients with atopic dermatitis, and incubation of keratinocytes with proinflammatory and type 2 cytokines upregulated B7-H6, leading to increased ILC2 cytokine production. NKp30-B7-H6 interaction is a novel cell contact mechanism that mediates activation of ILC2 and identifies a potential target for the development of novel therapeutics for atopic dermatitis and other atopic diseases.

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.

Gutowska-Owsiak D, Greenwald L, Watson C, Selvakumar TA, Wang X, Ogg GS. 2014. The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin. Br J Dermatol, 171 (4), pp. 771-778. | Show Abstract | Read more

BACKGROUND: Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. OBJECTIVES: To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. METHODS: Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. RESULTS: We detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-α, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. CONCLUSIONS: Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention.

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Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie ANJ, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells Journal of Allergy and Clinical Immunology, 133 (4), | Show Abstract | Read more

Background Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on T H2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. Objectives We sought to determine the role of PGD2 and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. Methods The effects of PGD 2, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD2 under physiologic conditions were evaluated by using the supernatant from activated mast cells. Results PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD 2 on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Conclusions PGD2 is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s. © 2013 American Academy of Allergy, Asthma & Immunology.

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.

Salimi M, Barlow JL, Saunders SP, Xue L, Gutowska-Owsiak D, Wang X, Huang LC, Johnson D et al. 2013. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J Exp Med, 210 (13), pp. 2939-2950. | Show Abstract | Read more

Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.

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.

Huang LC, Pan X, Yang H, Wan LK, Stewart-Jones G, Dorrell L, Ogg G. 2013. Linking genotype to phenotype on beads: high throughput selection of peptides with biological function. Sci Rep, 3 pp. 3030. | Show Abstract | Read more

Although peptides are well recognised biological molecules in vivo, their selection from libraries is challenging because of relative low affinity whilst in linear conformation. We hypothesized that multiplexed peptides and DNA on the surface of beads would provide a platform for enhanced avidity and the selection of relevant peptides from a library (ORBIT bead display). Using human immunodeficiency virus (HIV-1) gp120 as a target, we identify peptides that inhibit HIV-1 replication in vitro through blocking of protein:protein interaction with the co-receptor CCR5. The bead display approach has many potential applications for probing biological systems and for drug lead development.

Crack LR, Jones L, Malavige GN, Patel V, Ogg GS. 2012. Human antimicrobial peptides LL-37 and human β-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus. Clin Exp Dermatol, 37 (5), pp. 534-543. | Show Abstract | Read more

BACKGROUND: There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human β-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). AIM: To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human β-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. METHODS: Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. RESULTS: LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Pre-incubation of VZV with LL-37, but not hBD-2, further reduced VZV load. CONCLUSIONS: Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE.

Malavige GN, McGowan S, Atukorale V, Salimi M, Peelawatta M, Fernando N, Jayaratne SD, Ogg G. 2012. Identification of serotype-specific T cell responses to highly conserved regions of the dengue viruses. Clin Exp Immunol, 168 (2), pp. 215-223. | Show Abstract | Read more

Determining previous infecting dengue virus (DENV) serotypes has been difficult due to highly cross-reactive immune responses from previous DENV infections. Determining the correlates of serotype-specific immune responses would be crucial in understanding dengue transmission in the community and would also help to determine the correlates of protective immune responses. Therefore, we set out to define highly conserved, serotype-specific regions of the DENVs. Serotype-specific and highly conserved regions of the four DENV serotypes were identified using Basic Local Alignment Search Tool (BLAST) searches and custom perl scripts. Using ex-vivo and cultured enzyme-linked immunospot (ELISPOT) assays, we identified serotype-specific T cell epitopes within the four DENV serotypes in healthy adult donors from Sri Lanka. We identified T cell responses to 19 regions of the four DENV serotypes. Six peptides were from the NS2A region and four peptides were from the NS4A region. All immune donors responded to peptides of at least two DENV serotypes, suggesting that heterologous infection is common in Sri Lanka. Eight of 20 individuals responded to at least two peptides of DENV-4, despite this serotype not being implicated previously in any of the epidemics in Sri Lanka. The use of these regions to determine past and current infecting DENV serotypes will be of value to characterize further the dynamics of silent dengue transmission in the community. In addition, these T cell responses to these regions could be used to characterize DENV serotype-specific immune responses and thus possibly help us to understand the immune correlates of a protective immune response.

Gutowska-Owsiak D, Schaupp AL, Salimi M, Selvakumar TA, McPherson T, Taylor S, Ogg GS. 2012. IL-17 downregulates filaggrin and affects keratinocyte expression of genes associated with cellular adhesion. Exp Dermatol, 21 (2), pp. 104-110. | Show Abstract | Read more

Atopic eczema and psoriasis are common skin diseases. While it is well established that the pathogenesis of these diseases varies, both are characterized by impairment in epidermal barrier function and abnormal IL-17 expression in the skin and peripheral blood. Recent findings indicated that filaggrin is essential during barrier formation and its insufficiency underlies the pathogenesis of atopic eczema. Filaggrin downregulation has also been reported in psoriasis. It is clear that Th1/Th2 bias influences expression of the protein, but an analysis of the effects of interleukin-17 (IL-17) on the expression of the protein and profilaggrin-processing enzymes has not yet been reported. In addition, the effect of the cytokine on components of functional epidermal barrier, tight junctions and adhesion/desmosomal proteins, has not been elucidated. Keratinocytes were exposed to interleukin-17A, and microarray analysis was performed. Filaggrin protein level was assessed by western blot. We have observed a significant decrease in profilaggrin mRNA level in interleukin-17A-exposed cultures (P = 0.008). Expression of processing enzymes was also altered, indicating an indirect effect of the cytokine on filaggrin production/degradation. Moreover, expression of many genes involved in cellular adhesion was also decreased. A significant downregulation of filaggrin at the protein level was detected by western blot in immortal and primary keratinocytes. Gene ontology analysis indicated changes in keratinization, epidermal differentiation and formation of the cornified envelope. We conclude that IL-17A downregulates the expression of filaggrin and genes important for cellular adhesion which could affect epidermal barrier formation. This effect potentially contributes to barrier dysfunction and could become a possible therapeutic target.

Aslam A, Chan H, Warrell DA, Misbah S, Ogg GS. 2010. Tracking antigen-specific T-cells during clinical tolerance induction in humans. PLoS One, 5 (6), pp. e11028. | Show Abstract | Read more

Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen specific T-cell responses during immunotherapy can provide novel insights into mechanisms of tolerance induction in humans and identify new potential treatment targets.

Malavige GN, Jones L, Kamaladasa SD, Wijewickrama A, Seneviratne SL, Black AP, Ogg GS. 2008. Viral load, clinical disease severity and cellular immune responses in primary varicella zoster virus infection in Sri Lanka. PLoS One, 3 (11), pp. e3789. | Show Abstract | Read more

BACKGROUND: In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied. METHODOLOGY: We used IFNgamma ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection. PRINCIPAL FINDINGS: Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNgamma ELISpot responses of patients (P<0.001, r = -0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation. CONCLUSIONS: Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses.

Black AP, Ardern-Jones MR, Kasprowicz V, Bowness P, Jones L, Bailey AS, Ogg GS. 2007. Human keratinocyte induction of rapid effector function in antigen-specific memory CD4+ and CD8+ T cells. Eur J Immunol, 37 (6), pp. 1485-1493. | Show Abstract | Read more

The ability of human keratinocytes to present antigen to T cells is controversial and, indeed, it has been suggested that keratinocytes may promote T cell hyporesponsiveness. Furthermore, it is unclear whether keratinocytes can process antigen prior to MHC class I and class II presentation. We tested the ability of keratinocytes to induce functional responses in epitope-specific CD4+ and CD8+ memory T cells using peptides, protein and recombinant expression vectors as sources of antigen. Keratinocytes were able to efficiently process and present protein antigen to CD4+ T cells, resulting in cytokine secretion (Th1 and Th2). This interaction was dependent on keratinocyte expression of HLA class II and ICAM-1, which could be induced by IFN-gamma. In addition, keratinocytes could present virally encoded or exogenous peptide to CD8+ T cells, resulting in T cell cytokine production and target cell lysis. Finally, T cell lines grown using keratinocytes as stimulators showed no loss of function. These findings demonstrate that keratinocytes are able to efficiently process and present antigen to CD4+ and CD8+ memory T cells and induce functional responses. The findings have broad implications for the pathogenesis of cutaneous disease and for transcutaneous drug or vaccine delivery.

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