Detection and Characterizationof 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.     

Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses

Background

Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.

Methodology/Principal Findings

We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells.

Conclusions/Significance

The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.     

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.     

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.     

Enhancement of DNA vaccine potency through linkage of antigen gene to ER chaperone molecules,ER-60, tapasin,and calnexin

DNA vaccines have emerged as an attractive approach for generating antigen-specific immunotherapy. Strategies that enhance antigen presentation may potentially be used to enhance DNA vaccine potency. Previous experiments showed that chimeric DNA vaccines utilizing endoplasmic reticulum (ER) chaperone molecules, such as Calreticulin (CRT), linked to an antigen were capable of generating antigen-specific CD8⁺ T cell immune responses in vaccinated mice. In this study, we tested DNA vaccines encoding the ER chaperone molecules ER-60, tapasin (Tap), or calnexin (Cal), linked to human papillomavirus type 16 (HPV-16) E7 for their abilities to generate E7-specific T cell-mediated immune responses and antitumor effects in vaccinated mice. Our results demonstrated that vaccination with DNA encoding any of these chaperone molecules linked to E7 led to a significant increase in the frequency of E7-specific CD8⁺ T cell precursors and generated stronger antitumor effects against an E7-expressing tumor in vaccinated mice compared to vaccination with wild-type E7 DNA. Our data suggest that DNA vaccines employing these ER chaperone molecules linked to antigen may enhance antigen-specific CD8⁺ T cell immune responses, resulting in a significantly more potent DNA vaccine.     

Norovirus P Particle Efficiently Elicits Innate,Humoral and Cellular Immunity

Norovirus (NoV) P domain complexes, the 24 mer P particles and the P dimers, induced effective humoral immunity, but their role in the cellular immune responses remained unclear. We reported here a study on cellular immune responses of the two P domain complexes in comparison with the virus-like particle (VLP) of a GII.4 NoV (VA387) in mice. The P domain complexes induced significant central memory CD4⁺ T cell phenotypes (CD4⁺ CD44⁺ CD62L⁺ CCR7⁺) and activated polyclonal CD4⁺ T cells as shown by production of Interleukin (IL)-2, Interferon (IFN)-γ, and Tumor Necrosis Factor (TNF)-α. Most importantly, VA387-specific CD4⁺ T cell epitope induced a production of IFN-γ, indicating an antigen-specific CD4⁺ T cell response in P domain complex-immunized mice. Furthermore, P domain complexes efficiently induced bone marrow-derived dendritic cell (BMDC) maturation, evidenced by up-regulation of co-stimulatory and MHC class II molecules, as well as production of IL-12 and IL-1β. Finally, P domain complex-induced mature dendritic cells (DCs) elicited proliferation of specific CD4⁺ T cells targeting VA387 P domain. Overall, we conclude that the NoV P domain complexes are efficiently presented by DCs to elicit not only humoral but also cellular immune responses against NoVs. Since the P particle is highly effective for both humoral and cellular immune responses and easily produced in Escherichia coli (E. coli), it is a good choice of vaccine against NoVs and a vaccine platform against other diseases.     

CTLA-4 blockade increases antigen-specific CD8+ T cells in prevaccinated patients with melanoma: three cases

Background

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.

Methods

To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100^209?C217 and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100^209?C217 (ITDQVPFSV), tyrosinase^369?C377 (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.

Results

Following vaccination, patients generated weak to no CD4⁺ or CD8⁺ T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7^loCD45RA^lo) tetramer⁺CD8⁺ T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.

Conclusion

Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially ??boosting?? the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.     

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

Soluble MHC–peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8⁺ T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens. A. I. Dodi and P. J. Travers contributed equally to this work. This paper is an original contribution from the meeting which took place on 28 and 29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. I. A. “Tony” Dodi (^†29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

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.     

MHC class I multimers

T lymphocytes play a key role in the immune response to both foreign and self peptide antigens, which they recognize in combination with MHC molecules. In the past it has been difficult to analyse objectively the specificity, frequency and intensity of T cell responses. The recent application of fluorescent-labelled MHC class I multimers, however, has provided a powerful experimental approach to the direct visualisation of antigen-specific T cells. As a result, our perspective of how T cells respond to both viruses and other antigens in vivo has been greatly enhanced.     

Brucella abortus induces intracellular retention of MHC‐I molecules in human macrophages down‐modulating cytotoxic CD8+ T cell responses

Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8⁺ T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN‐γ‐induced MHC‐I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC‐I down‐modulation correlated with the development of diminished CD8⁺ cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8⁺ T lymphocytes and a diminished percentage of IFN‐γ‐producing CD8⁺ T cells. Inhibition of MHC‐I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC‐I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC‐I molecules whereby B. abortus would avoid CD8⁺ cytotoxic T cell responses, evading their immunological surveillance.     

Enhanced immune response induced by P5 HER2/neu‐derived peptide‐pulsed dendritic cells as a preventive cancer vaccine

Dendritic cells are special and powerful antigen‐presenting cells that can induce primary immune responses against tumour‐associated antigens. They can present antigens via both MHC‐I and MHC‐II, so they have the ability to stimulate both cytotoxic T lymphocytes and T helper cells. Furthermore, CD8⁺ cytotoxic T lymphocytes require activation by CD4⁺ T cells. This requires a CD4⁺T cell activator molecule, of which PADRE is one of the best. We chose an approach to use both of these important arms of the immune system. We prepared dendritic cells from mouse bone marrow, loaded them with our target peptides (P5 peptide alone or P5 + PADRE), and then injected these pulsed dendritic cells alone or in combination with CpG‐ODN (as adjuvant) into BALB/C mice. After the last boosting dose, mice were inoculated with TUBO cells, which overexpress HER2/neu. Two weeks after the tumour cell injection, immunological tests were performed on splenocyte suspensions, and the remaining mice were evaluated for tumour growth and survival. Our data indicate the formulation that contains PADRE plus P5 loaded onto DC in combination with CpG‐ODN was the most effective formulation at inducing immune responses. Interferon production in CD4⁺ and CD8⁺ gated cells, cytotoxicity rates of target cells and mice survival were all significantly greater in this group than in controls, and all the mice in this group were tumour‐free throughout the experiment. Based on our results and the role of HER2/neu as a candidate in human immunotherapy, this approach may be an effective cancer treatment.     

Characterization of human CD4 helper T cell responses against Aurora kinase A

Aurora kinase A (Aurora-A) is a cell cycle-associated serine–threonine kinase that is overexpressed by various types of cancer and is highly associated with poor prognosis. Since the expression of Aurora-A in normal tissues has been shown to be significantly lower as compared to tumor cells, this protein is being considered as a potential tumor-associated antigen for developing immunotherapies. The goal in the present study was to identify CD4 helper T lymphocyte (HTL) epitopes for Aurora-A for the design of T cell-based immunotherapies against Aurora-A-expressing tumors. Synthetic peptides corresponding to potential HTL epitopes were identified from Aurora-A and used to stimulate CD4 T lymphocytes in vitro to generate antigen-specific HTL clones that were evaluated for antigen specificity, MHC restriction and for their ability to interact with Aurora-A-expressing tumor cells. The results show that two peptides (Aurora-A_161–175 and Aurora-A_233–247) were effective in generating HTL responses that were restricted by more than one MHC class II allele (i.e., promiscuous responses). The CD4 HTL clones were able to directly recognize Aurora-A-expressing tumor cells in an antigen-specific and MHC class II-restricted manner and some of the clones displayed cytolytic activity toward Aurora-A + tumor cells. Both of these peptides were capable of stimulating in vitro T cell responses in patients with bladder cancer.     

Enhanced Cross-Presentation and Improved CD8+ T Cell Responses after Mannosylation of Synthetic Long Peptides in Mice

The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8⁺ T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response in vitro and in vivo and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.     

Single chamber microbial fuel cell with spiral anode for dairy wastewater treatment

The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individual's medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individual's response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities were used to detect and characterize CD4(+) T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabeled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.     

Characterization of MHC class-I restricted TCRαβ<Superscript>+</Superscript> CD4<Superscript>−</Superscript> CD8<Superscript>−</Superscript> double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination

The immune attack against malignant tumors require the concerted action of CD8⁺ cytotoxic T lymphocytes (CTL) as well as CD4⁺ T helper cells. The contribution of T cell receptor (TCR) αβ⁺ CD4⁻ CD8⁻ double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2⁺ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo. A. Mackensen and K. Fischer contributed equally to this work and should be considered joint senior authors. This work was supported by the Deutsche Forschungsgemeinschaft (MA 1351/5-1, KFO 146) and NIH grants CA90873, CA102280, 104947 (MIN). Companion paper: “Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition” by Tamson V. Moore et al. doi: .     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

Increased Synapse Formation Obtained by T Cell Epitopes Containing a CxxC Motif in Flanking Residues Convert CD4+ T Cells into Cytolytic Effectors

The nature of MHC class II-binding epitopes not only determines the specificity of T cell responses, but may also alter effector cell functions. Cytolytic CD4+ T cells have been observed primarily in anti-viral responses, but very little is known about the conditions under which they can be elicited. Their potential as regulators of immune responses, however, deserves investigations. We describe here that inclusion of a thiol-disulfide oxidoreductase motif within flanking residues of class II-restricted epitopes results, both in vitro and in vivo, in elicitation of antigen-specific cytolytic CD4+ T cells through increased synapse formation. We show that both naïve and polarized CD4+ T cells, including Th17 cells, can be converted by cognate recognition of such modified epitopes. Cytolytic CD4+ T cells induce apoptosis on APCs by Fas-FasL interaction. These findings potentially open the way towards a novel form of antigen-specific immunosuppression.     

MHC class I multimers

T lymphocytes play a key role in the immune response to both foreign and self peptide antigens, which they recognize in combination with MHC molecules. In the past it has been difficult to analyse objectively the specificity, frequency and intensity of T cell responses. The recent application of fluorescent-labelled MHC class I multimers, however, has provided a powerful experimental approach to the direct visualisation of antigen-specific T cells. As a result, our perspective of how T cells respond to both viruses and other antigens in vivo has been greatly enhanced.