Detection and characterization of cellular immune responses using peptide-MHC microarrays

The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease. We have developed and tested a method that uses arrays of peptide–MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells. CD4⁺ or CD8⁺ lymphocytes can be captured in accordance with their ligand specificity using an array of peptide–MHC complexes printed on a film-coated glass surface. We have characterized the specificity and sensitivity of a peptide–MHC array using labeled lymphocytes from T cell receptor transgenic mice. In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse. This approach should be useful for epitope discovery, as well as for characterization and analysis of multiple epitope-specific T cell populations during immune responses associated with viral and bacterial infection, cancer, autoimmunity, and vaccination.     

Localized Populations of CD8low/? MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium

CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8⁺. Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer⁺CD8^low/− cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer⁺CD20⁻ cells inside B cell follicles and that tetramer⁺ cells did not stain with γδ-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer⁺CD8^low/− cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium.     

Clonal focusing of epitope-specific CD8+ T lymphocytes in rhesus monkeys following vaccination and simian-human immunodeficiency virus challenge

To afford the greatest possible immune protection, candidate human immunodeficiency virus (HIV) vaccines must generate diverse and long-lasting CD8⁺ T lymphocyte responses. In the present study, we evaluate T-cell receptor Vβ (variable region beta) gene usage and a CDR3 (complementarity-determining region 3) sequence to assess the clonality of epitope-specific CD8⁺ T lymphocytes generated in rhesus monkeys following vaccination and simian-human immunodeficiency virus (SHIV) challenge. We found that vaccine-elicited epitope-specific CD8⁺ T lymphocytes have a clonal diversity comparable to those cells generated in response to SHIV infection. Moreover, we show that the clonal diversity of vaccine-elicited CD8⁺ T-lymphocyte responses is dictated by the epitope sequence and is not affected by the mode of antigen delivery to the immune system. Clonal CD8⁺ T-lymphocyte populations persisted following boosting with different vectors, and these clonal cell populations could be detected for as long as 4 years after SHIV challenge. Finally, we show that the breadth of these epitope-specific T lymphocytes transiently focuses in response to intense SHIV replication. These observations demonstrate the importance of the initial immune response to SHIV, induced by vaccination or generated during primary infection, in determining the clonal diversity of cell-mediated immune responses and highlight the focusing of this clonal diversity in the setting of high viral loads. Circumventing this restricted CD8⁺ T-lymphocyte clonal diversity may present a significant challenge in the development of an effective HIV vaccine strategy.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

Efficient Generation of Mucosal and Systemic Antigen-Specific CD8+ T-Cell Responses following Pulmonary DNA Immunization

Although mucosal CD8⁺ T-cell responses are important in combating mucosal infections, the generation of such immune responses by vaccination remains problematic. In the present study, we evaluated the ability of plasmid DNA to induce local and systemic antigen-specific CD8⁺ T-cell responses after pulmonary administration. We show that the pulmonary delivery of plasmid DNA formulated with polyethyleneimine (PEI-DNA) induced robust systemic CD8⁺ T-cell responses that were comparable in magnitude to those generated by intramuscular (i.m.) immunization. Most importantly, we observed that the pulmonary delivery of PEI-DNA elicited a 10-fold-greater antigen-specific CD8⁺ T-cell response in lungs and draining lymph nodes of mice than that of i.m. immunization. The functional evaluation of these pulmonary CD8⁺ T cells revealed that they produced type I cytokines, and pulmonary immunization with PEI-DNA induced lung-associated antigen-specific CD4⁺ T cells that produced higher levels of interleukin-2 than those induced by i.m. immunization. Pulmonary PEI-DNA immunization also induced CD8⁺ T-cell responses in the gut and vaginal mucosa. Finally, pulmonary, but not i.m., plasmid DNA vaccination protected mice from a lethal recombinant vaccinia virus challenge. These findings suggest that pulmonary PEI-DNA immunization might be a useful approach for immunizing against pulmonary pathogens and might also protect against infections initiated at other mucosal sites.Since establishing that antigen-specific CD8⁺ T-cell populations in mucosal sites may confer protection against intracellular pathogens that initiate infections at mucosal surfaces, vaccine strategies have been explored for eliciting cellular immune responses in mucosal tissues (40). Studies have been done to evaluate the immunogenicity of vaccines delivered to a variety of mucosal surfaces, including those of the nose, intestine, rectum, and vagina. These studies have shown that immunization at mucosal sites can induce larger numbers of antigen-specific CD8⁺ T cells in mucosal tissues than parenteral immunization (3).Particular attention has focused on the lungs as a target for mucosal immunization. The lungs are an important mucosal portal of entry for pathogens. They are also a readily accessed mucosal site for the delivery of immunogens that might induce diverse mucosal immune responses. Pulmonary immunization strategies have been shown to generate potent Th1 responses and protective immunity against respiratory challenge with pathogens in several animal models (4, 29, 32, 37, 38).Because of the ease of generating vaccine constructs and the ability to administer repeated inoculations of the same vector, DNA immunization remains a promising vaccination strategy for eliciting cellular immune responses. Only a limited number of studies have been done to evaluate the immunogenicity of DNA vaccines following pulmonary delivery (4, 32). Although the importance of CD8⁺ T lymphocytes in eradicating mucosal infections has been well established, it has not been determined whether pulmonary DNA immunization can induce robust functional CD8⁺ T-cell responses.In the present study, we characterized antigen-specific CD8⁺ T lymphocytes in mice induced by the noninvasive pulmonary administration of plasmid DNA complexed to the cationic polymer polyethyleneimine (PEI). We demonstrate that the delivery of a DNA vaccine to the airways can induce a high frequency of functional antigen-specific CD8⁺ T cells in both systemic and mucosal sites.     

Manipulating IL-2 Availability Amid Presentation of Donor MHC Antigens Suppresses Murine Alloimmune Responses by Inducing Regulatory T Cells

Background

Major histocompatibility complex (MHC) antigens are important for alloimmune responses as well as immune tolerance. Previous studies have shown that presentation of donor MHC antigens by donor-specific transfusion prior to or upon transplantation promotes transplant tolerance induced by other agents. However, it is unclear whether presentation of donor MHC antigens by DNA vaccination induces long-term allograft survival.

Methodology/Principal Findings

We investigated whether presentation of MHC class-II and/or class-I donor antigens by DNA vaccination suppresses alloimmune responses and promotes long-term allograft acceptance. We initially found that presentation of both MHC donor antigens by DNA vaccination itself prior to transplantation fails to significantly prolong islet allograft survival in otherwise untreated mice. However, islet allograft survival was significantly prolonged when MHC class-II DNA vaccination was accompanied with IL-2 administration (MHCII + IL-2) while MHC class-I DNA vaccination was followed by IL-2 and subsequent neutralizing anti-IL-2 treatments (MHCI + IL-2/anti-IL-2). Especially, this protocol promoted long-term allograft survival in the majority of recipients (57%) when combined with low doses of rapamycin post-transplantation. Importantly, MHCII + IL-2 induced FoxP3+ Treg cells in both spleens and grafts and suppressed graft-infiltrating CD4+ cell proliferation, whereas MHCI + IL-2/anti-IL-2 mainly inhibited graft-infiltrating CD8+ cell proliferation and donor-specific CTL activity. The combined protocol plus rapamycin treatment further reduced both CD4+ and CD8+ T cell proliferation as well as donor-specific CTL activity but spared FoxP3+ Treg cells. Depleting CD25+ Treg cells or adoptive transfer of pre-sensitized CD8+ T cells abolished this long-term allograft survival.

Conclusions/Significance

Manipulating IL-2 availability during presentation of MHC class-II and class-I donor antigens by DNA vaccination pre-transplantation induces Treg cells, suppresses alloimmune responses and promotes long-term allograft survival.     

Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

Background

Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered.

Principal Findings

Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with ‘MHC-loading enhancer’ (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE.

Conclusion

MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy.     

Vaccine-Induced,Pseudorabies Virus-Specific,Extrathymic CD4+CD8+ Memory T-Helper Cells in Swine

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4⁺CD8⁺ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4⁺CD8⁺ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4⁺CD8⁺ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4⁺CD8⁺ memory T lymphocytes and showed that CD4⁺CD8⁺ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4⁺CD8⁺ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, is the causative agent of Aujeszky’s disease. This disease is lethal to young pigs and causes important economic losses (52). Therefore, vaccination of pigs is practiced in many countries.Several humoral immune system effector mechanisms are involved in the protection of pigs from PRV infection. Virus-neutralizing antibodies, antibodies mediating antibody-dependent cell-mediated cytotoxicity, and antibodies mediating complement-mediated lysis of PRV-infected target cells have been demonstrated (22, 23, 53, 54). The main targets of this humoral immune response were shown to be the viral glycoproteins (3, 45), and passive immunization with monoclonal antibodies (MAbs) against gB, gC, and gD protects pigs from a lethal challenge (20, 49).The protection conferred through cell-mediated immunity is poorly understood. An increase in major histocompatibility complex (MHC)-unrestricted cell-mediated cytotoxicity against uninfected and PRV-infected cells has been detected after infection or vaccination of pigs with PRV (16, 53, 54), and specific cellular immune responses to PRV infections could be demonstrated by stimulation of proliferation and lymphokine secretion of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as well as by the detection of PRV-specific cytotoxic lymphocytes (21, 56).There are some difficulties in defining more precisely the impact of cell-mediated immune effector mechanisms to protection from PRV-infection and their interplay with the observed humoral immune response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and mice. The pig has a substantial number of CD4⁻CD8⁻ T lymphocytes in the peripheral blood (4, 6, 12, 36, 39). In young animals, this subpopulation of T lymphocytes comprises up to 60% of the T lymphocytes and contains mainly γδ T lymphocytes. The pig is also the only species so far known to contain a substantial number of resting extrathymic CD4⁺CD8⁺ T lymphocytes (28, 36, 39). This T-lymphocyte population shows morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that CD4⁺CD8⁺ T lymphocytes comprise memory T cells which proliferate upon stimulation with recall antigen (43, 55). Since the observed proliferative response was shown to be MHC class II-restricted, it was speculated that the porcine CD4⁺CD8⁺ T-cell subset contains memory T-helper lymphocytes (43). However, the ability of these T lymphocytes to secrete cytokines or to provide help to B cells has so far not been demonstrated.To gain a better understanding of immune effector mechanisms conferring protection from PRV infection, the function of these unusual extrathymic T-lymphocyte subsets has to be elucidated. In the present study, we identified two T-cell epitopes on glycoprotein gC which are primed during vaccination of d/d haplotype inbred pigs (41) against PRV and demonstrated that MHC class II-restricted, peripheral CD4⁺CD8⁺ memory T lymphocytes are the responding T lymphocytes. We were further able to show that PRV-specific, extrathymic CD4⁺CD8⁺ T lymphocytes are able to secrete cytokines and have the capacity to stimulate the secretion of PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results demonstrate that porcine CD4⁺CD8⁺ T lymphocytes can function as memory T-helper cells and can direct humoral anti-PRV memory responses.     

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8⁺ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8⁺ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8⁺ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8⁺ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8⁺ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8⁺ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8⁺ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.     

Self-Organized Criticality Theory of Autoimmunity

Background

The cause of autoimmunity, which is unknown, is investigated from a different angle, i.e., the defect in immune ‘system’, to explain the cause of autoimmunity.

Methodology/Principal Findings

Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4⁺ T cells led to the development of autoantibody-inducing CD4⁺ T (aiCD4⁺ T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4⁺ T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8⁺ T cells, driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE).

Conclusions/Significance

Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host''s immune ‘system’ by repeated immunization with antigen, to the levels that surpass system''s self-organized criticality.     

rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector

Background

BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals).

Methods and Findings

Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4⁺, CD8alpha/beta⁺, and CD8alpha/alpha⁺ T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha⁺ T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha⁺ T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity.

Conclusion

AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials     

A Comparative Approach Linking Molecular Dynamics of Altered Peptide Ligands and MHC with In Vivo Immune Responses

Background

The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood.

Results

We analyzed the immunological consequences of the interaction of MHC class II (I-A^u) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4⁺ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation.     

Glutamic Acid Decarboxylase-Derived Epitopes with Specific Domains Expand CD4+CD25+ Regulatory T Cells

Background

CD4⁺CD25⁺ regulatory T cell (Treg)-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used.

Methodology/Principal Findings

Splenic lymphocytes from nonobese diabetic (NOD) mice were stimulated with different glutamic acid decarboxylase (GAD)-derived epitopes for 7–10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524–538)-expanded CD4⁺CD25⁺ T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509–528)- or p530 (GAD530–543)-expanded CD4⁺CD25⁺ T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes) between the above-mentioned epitopes and MHC class II I-A^g7 were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A^g7, while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A^g7. Furthermore, p524 bound to I-A^g7 more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570–585, p570), which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4⁺CD25⁺ T cells suppressed the onset of diabetes in NOD mice.

Conclusions/Significance

These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of protective epitopes to expand functional Tregs.     

Regulatory T Cells Suppress Natural Killer Cells during Plasmid DNA Vaccination in Mice,Blunting the CD8+ T Cell Immune Response by the Cytokine TGFβ

Background

CD4⁺CD25⁺ regulatory T cells (Tregs) suppress adaptive T cell-mediated immune responses to self- and foreign-antigens. Tregs may also suppress early innate immune responses to vaccine antigens and might decrease vaccine efficacy. NK and NKT cells are the first responders after plasmid DNA vaccination and are found at the site of inoculation. Earlier reports demonstrated that NKT cells could improve plasmid DNA efficacy, a phenomenon not found for NK cells. In fact, it has been shown that under certain disease conditions, NK cells are suppressed by Tregs via their release of IL-10 and/or TGFβ. Therefore, we tested the hypothesis that NK cell function is suppressed by Tregs in the setting of plasmid DNA vaccination.

Methodology/Principal Findings

In this study we show that Tregs directly inhibit NK cell function during plasmid DNA vaccination by suppressing the potentially 10-fold, NK cell-mediated, augmentation of plasmid DNA antigen-specific CD8⁺ T cells. We found that this phenomenon is dependent on the secretion of cytokine TGFβ by Tregs, and independent of IL-10.

Conclusions

Our data indicate a crucial function for Tregs in blocking plasmid DNA vaccine-elicited immune responses, revealing potentially novel strategies for improving the efficiency of plasmid DNA vaccines including chemical- or antibody-induced localized blockage of Treg-mediated suppression of NK cells at the site of plasmid DNA vaccine inoculation.     

Mucosal Trafficking of Vector-Specific CD4+ T Lymphocytes following Vaccination of Rhesus Monkeys with Adenovirus Serotype 5

Post hoc analysis of the phase 2b Step study evaluating a recombinant adenovirus serotype 5 (rAd5)-based HIV-1 vaccine candidate suggested a potential increased risk of HIV-1 acquisition in subjects who were baseline Ad5 seropositive and uncircumcised. These concerns had a profound impact on the HIV-1 vaccine development field, although the mechanism underlying this observation remains unknown. It has been hypothesized that rAd5 vaccination of baseline Ad5-seropositive individuals may have resulted in anamnestic, vector-specific CD4⁺ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Here we show that Ad5-specific CD4⁺ T lymphocyte responses at mucosal sites following rAd5-Gag/Pol/Nef vaccination were comparable in rhesus monkeys with and without baseline Ad5 immunity. Moreover, the total cellular inflammatory infiltrates and the CD3⁺, CD4⁺, HLA-DR⁺, Ki67⁺, and langerin⁺ cellular subpopulations in colorectal and foreskin mucosa were similar in both groups. Thus, no greater trafficking of Ad5-specific CD4⁺ T lymphocytes to mucosal target sites was observed following rAd5 vaccination of rhesus monkeys with baseline Ad5 immunity. These findings from this nonhuman primate model provide evidence against the hypothesis that recruitment of vector-specific target cells to mucosal sites led to increased HIV-1 acquisition in Ad5-seropositive, uncircumcised vaccinees in the Step study.The Step study revealed a potential increased risk of HIV-1 acquisition among adenovirus serotype 5 (Ad5)-seropositive, uncircumcised subjects who received the Merck recombinant Ad5 (rAd5)-Gag/Pol/Nef vaccine candidate (2, 6). It has been hypothesized that rAd5 vaccination of Ad5-seropositive individuals may have resulted in robust expansion and activation of vector-specific CD4⁺ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Our laboratory and others have recently demonstrated that total and vector-specific CD4⁺ T lymphocytes in peripheral blood in Ad5-seropositive volunteers were comparable to or lower than the levels in Ad5-seronegative volunteers following rAd5-Gag vaccination in the Merck phase 1 studies (4, 8). However, mucosal biopsy specimens were not obtained in these clinical trials, and thus the extent of inflammatory infiltrates and vector-specific CD4⁺ T lymphocytes in colorectal and foreskin mucosa could not be evaluated in these prior studies.It has also recently been reported that vector-specific CD4⁺ T lymphocytes may upregulate mucosal homing integrin expression following exposure to Ad5 in short-term in vitro cultures (1). These findings highlight the importance of directly investigating the extent and nature of vector-specific CD4⁺ T lymphocytes at mucosal sites following rAd5 vaccination. Given the lack of mucosal biopsy samples from human subjects in the Step study, we developed a nonhuman primate model of preexisting adenovirus immunity to evaluate the extent and nature of inflammatory cell populations at mucosal sites following rAd5 vaccination.     

Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific,Autoreactive CD4 T Cells In Situ

We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139–151; the sections were costained with IA^s/PLP 139–151 (specific) or Theiler''s murine encephalomyelitis virus (TMEV) 70–86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139–151 but not for TMEV 70–86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139–151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigen-specific T cells by counting the number of dextramer⁺ CD4⁺ T cells in the ‘Z’ serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-α (Myhc) 334–352. Heart sections prepared from immunized mice were costained with Myhc 334–352 (specific) or bovine ribonuclease 43–56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.     

The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critically Involved in the Development of Cytotoxic CD8+ T Cells in Mice and Humans

Background

The neuropeptide alpha-melanocyte-stimulating hormone is well known as a mediator of skin pigmentation. More recently, it has been shown that alpha-melanocyte-stimulating hormone also plays pivotal roles in energy homeostasis, sexual function, and inflammation or immunomodulation. Alpha-melanocyte-stimulating hormone exerts its antiinflammatory and immunomodulatory effects by binding to the melanocortin-1 receptor, and since T cells are important effectors during immune responses, we investigated the effects of alpha-melanocyte-stimulating hormone on T cell function.

Methodology/Principal Findings

T cells were treated with alpha-melanocyte-stimulating hormone, and subsequently, their phenotype and function was analyzed in a contact allergy as well as a melanoma model. Furthermore, the relevance of alpha-melanocyte-stimulating hormone–mediated signaling for the induction of cytotoxicity was assessed in CD8⁺ T cells from melanoma patients with functional and nonfunctional melanocortin-1 receptors. Here we demonstrate that the melanocortin-1 receptor is expressed by murine as well as human CD8⁺ T cells, and we furthermore show that alpha-melanocyte-stimulating hormone/melanocortin-1 receptor–mediated signaling is critical for the induction of cytotoxicity in human and murine CD8⁺ T cells. Upon adoptive transfer, alpha-melanocyte-stimulating hormone–treated murine CD8⁺ T cells significantly reduced contact allergy responses in recipient mice. Additionally, the presented data indicate that alpha-melanocyte-stimulating hormone via signaling through a functional melanocortin-1 receptor augmented antitumoral immunity by up-regulating the expression of cytotoxic genes and enhancing the cytolytic activity in tumor-specific CD8⁺ T cells.

Conclusions/Significance

Together, these results point to an important role of alpha-melanocyte-stimulating hormone in MHC class I-restricted cytotoxicity. Therefore, treatment of contact allergies or skin cancer with alpha-melanocyte-stimulating hormone or other more stable agonists of melanocortin-1 receptor might ameliorate disease or improve antitumoral immune responses.     

T Cell Detection of a B-Cell Tropic Virus Infection: Newly-Synthesised versus Mature Viral Proteins as Antigen Sources for CD4 and CD8 Epitope Display

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8⁺ and CD4⁺ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8⁺ and CD4⁺ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36–48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4⁺ T cells.     

Failure of Effector Function of Human CD8+ T Cells in NOD/SCID/JAK3?/? Immunodeficient Mice Transplanted with Human CD34+ Hematopoietic Stem Cells

Humanized mice, which are generated by transplanting human CD34⁺ hematopoietic stem cells into immunodeficient mice, are expected to be useful for the research on human immune responses. It is reported that antigen-specific T cell responses occur in immunodeficient mice transplanted with both human fetal thymus/liver tissues and CD34⁺ fetal cells, but it remains unclear whether antigen-specific T cell responses occur in those transplanted with only human CD34⁺ hematopoietic stem cells (HSCs). Here we investigated the differentiation and function of human CD8⁺ T cells reconstituted in NOD/SCID/Jak3^−/− mice transplanted with human CD34⁺ HSCs (hNOK mice). Multicolor flow cytometric analysis demonstrated that human CD8⁺ T cells generated from the CD34⁺ HSCs comprised only 3 subtypes, i.e., CD27^highCD28⁺CD45RA⁺CCR7⁺, CD27⁺CD28⁺CD45RA⁻CCR7⁺, and CD27⁺CD28⁺CD45RA⁻CCR7⁻ and had 3 phenotypes for 3 lytic molecules, i.e., perforin(Per)⁻granzymeA(GraA)⁻granzymeB(GraB)⁻, Per⁻GraA⁺GraB⁻, and Per^lowGraA⁺GraB⁺. These CD8⁺ T cells failed to produce IFN-γ and to proliferate after stimulation with alloantigens. These results indicate that the antigen-specific T cell response cannot be elicited in mice transplanted with only human CD34⁺ HSCs, because the T cells fail to develop normally in such mice.