Tetramer-guided Epitope Mapping Research Articles

Abstract B23: Enhanced HLA-II epitope prediction for immunotherapy with novel proteomics and genomics approaches

Abstract While tumor-reactive CD4+ T cells have been associated with effective immunotherapy responses, accurate prediction of MHC class II-restricted ligands remains a challenge, limiting our ability to harness CD4+ immunity for cancer therapy. To this end, we have developed a state-of-the-art MHC class II binding predictor, neonmhc2, trained on HLA-II ligands identified from mass spectrometry (MS) of cell lines engineered to express a single affinity-tagged HLA-II allele. Over 60 HLA-II alleles have been characterized to date. We demonstrate that neonmhc2 outperforms NetMHCIIpan, the current benchmark for class II prediction, in distinguishing 1) HLA-II ligands presented in cell lines and tissues, and critically, 2) immunogenic CD4+ epitopes identified with tetramer-guided epitope mapping. Furthermore, we show that numerous neoantigen peptides that were ranked highly by neonmhc2 but poorly by NetMHCIIpan were immunogenic in an ex vivo induction. We next studied HLA-II antigen processing in order to further boost our ability to predict CD4+ epitopes. We determined a gene-level bias by comparing transcript expression and gene length to the frequency of observations in MS, finding that secreted genes are over-represented in HLA-II ligandomes from tissues. We built numerous primary sequence-based processability predictors trained on MS data, but only achieved significant prediction improvement when using the simple feature of determining if a candidate peptide contained sequence that overlapped a previously observed HLA-II ligand. By combining neonmhc2 binding prediction, transcript expression, gene bias, and the overlap feature in an integrated presentation predictor, we were able to achieve up to a 61-fold increase in ability to predict HLA-II peptides presented from tissue MS data over NetMHCIIpan alone. We also sought to understand which cells are presenting HLA-II ligands in the tumor microenvironment to elucidate the presentation pathway most relevant to immunotherapy. By leveraging publicly available RNA-seq (bulk and single cell), we found that professional antigen-presenting cells rather than tumor cells are primarily responsible for HLA-II presentation. We developed a novel SILAC-based MS workflow to directly interrogate peptides derived from phagocytosed tumor cells that are presented by dendritic cells. The experiment revealed that mitochondrial genes are preferentially presented from phagocytosed cells. In conclusion, by integrating proteomics and genomics data at large scale, we have defined new rules for understanding HLA-II processing and presentation, particularly in the context of the tumor microenvironment. This work should enhance our ability to predict CD4+ epitopes for immunotherapy. Citation Format: Dewi Harjanto, Jennifer G. Abelin, Matthew Malloy, Prerna Suri, Tyler Colson, Scott P. Goulding, Amanda L. Creech, Lia R. Serrano, Gibbs Nasir, Yusuf Nasrullah, Christopher D. McGann, Diana Velez, Ying S. Ting, Asaf Poran, Daniel A. Rothenberg, Sagar Chhangawala, Alex Rubinsteyn, Jeff Hammerbacher, Richard B. Gaynor, Edward F. Fritsch, Rob C. Oslund, Dominik Barthelme, Terri A. Addona, Christina M. Arieta, Michael S. Rooney. Enhanced HLA-II epitope prediction for immunotherapy with novel proteomics and genomics approaches [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B23.

Tetramer-Guided Epitope Mapping: A Rapid Approach to Identify HLA-Restricted T-Cell Epitopes from Composite Allergens.

Tetramer-guided epitope mapping (TGEM) is a technique in immunology that permits the rapid identification of allergenic epitopes through peptide screening procedures utilizing human lymphocyte antigen (HLA) class II tetramers as staining reagents for detection. The identification of allergenic epitopes is a prerequisite for the accurate characterization of allergen-specific CD4+ T cells without in vitro stimulation. Additionally, these MHC-II/peptide complexes that interact with T-cell receptors (TCR) of pathogenic CD4+ T cells are compatible with a different number of assays like Intracelullar Cytokine Staining (ICS), and Carboxyfluorescein succinimidyl ester (CFSE) making it a robust technology to study the functionality of allergen-specific CD4+ T cells.

Circulating Memory CD4+ T Cells Target Conserved Epitopes of Rhinovirus Capsid Proteins and Respond Rapidly to Experimental Infection in Humans.

Rhinovirus (RV) is a major cause of common cold and an important trigger of acute episodes of chronic lung diseases. Antigenic variation across the numerous RV strains results in frequent infections and a lack of durable cross-protection. Because the nature of human CD4+ T cells that target RV is largely unknown, T cell epitopes of RV capsid proteins were analyzed, and cognate T cells were characterized in healthy subjects and those infected by intranasal challenge. Peptide epitopes of the RV-A16 capsid proteins VP1 and VP2 were identified by peptide/MHC class II tetramer-guided epitope mapping, validated by direct ex vivo enumeration, and interrogated using a variety of in silico methods. Among noninfected subjects, those circulating RV-A16-specific CD4+ T cells detected at the highest frequencies targeted 10 unique epitopes that bound to diverse HLA-DR molecules. T cell epitopes localized to conserved molecular regions of biological significance to the virus were enriched for HLA class I and II binding motifs, and constituted both species-specific (RV-A) and pan-species (RV-A, -B, and -C) varieties. Circulating epitope-specific T cells comprised both memory Th1 and T follicular helper cells, and were rapidly expanded and activated after intranasal challenge with RV-A16. Cross-reactivity was evidenced by identification of a common *0401-restricted epitope for RV-A16 and RV-A39 by tetramer-guided epitope mapping and the ability for RV-A16-specific Th1 cells to proliferate in response to their RV-A39 peptide counterpart. The preferential persistence of high-frequency RV-specific memory Th1 cells that recognize a limited set of conserved epitopes likely arises from iterative priming by previous exposures to different RV strains.

Longitudinal study of CD4 T cell responses following Japanese encephalitis virus vaccination (VIR2P.1165)

Abstract Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. There are currently a number of vaccines shown to effectively prevent disease. In the United States, an alum adsorbed vaccine, which consists of a formaldehyde-inactivated, attenuated JEV strain is available. Vaccine protection has been shown to be mediated by neutralizing antibody responses. However, the CD4 T cell response associated with vaccine protection has not been fully characterized. In particular, the JEV model also presents an opportunity to study the kinetics of T cell responses elicited by a multi-dose vaccination in human subjects. In this study, we characterized the longitudinal response of JEV-specific CD4 T cells following vaccination using tetramer guided epitope mapping (TGEM), direct ex vivo MHC class II tetramer staining, and intracellular cytokine staining. A total of 67 epitopes restricted by 7 common HLA-DRB1 alleles and one DRB5 allele were identified. Vaccination elicited a robust CD4 T cell response, which was substantially boosted after the second dose of vaccine was administered. JEV-specific CD4 T cells exhibited a memory phenotype and expressed CCR4 and CXCR3. Consistent with this surface phenotype, JEV specific responses were polyfunctional, secreting IFN-g, IL-4 and sometimes IL-17a. These results indicate that JEV vaccination induces a robust, polyfunctional CD4 T cell response that is similar in magnitude to other highly effective vaccines.

Vicilin and 11s globulin cashew allergens share promiscous CD4+ T cell epitopes with other tree nuts (APP2P.100)

Abstract Allergic reactions to cashew can be life threatening. While IgE-specific cashew epitopes have been studied, CD4+ T-cell specific epitopes for cashew remain uncharacterized. Additionally, cross-reactivity amongst tree nuts has not been examined in humans at the T-cell level. We sought to provide a thorough phenotypic analysis of cashew reactive T-cells in allergic and non-allergic subjects as well as to identify promiscuous epitopes among tree nut allergens. CD4+ T-cell responses by CD154 up-regulation assays indicate that cashew allergens Ana o 1 and Ana o 2 play a major role in cashew allergy. Multiple Ana o 1 and Ana o 2 T-cell epitopes were identified with the Tetramer-Guided Epitope Mapping approach. Direct ex vivo staining showed that the majority of Ana o 1 and Ana o 2-specific T-cells have a CCR4+ TCM (central memory) phenotype while a subset of these T-cells express CCR4+CCR6+ in allergic subjects. Conversely, in non-allergic subjects a CXCR3+ population was observed. Cashew-specific T-cell clones for several epitope regions responded robustly to hazelnut and pistachio but not to walnut, while some epitopes responded to cashew exclusively and were found to be species specific. Allergic responses to cashew could be triggered by cross-reactive tree nut epitopes, therefore, restricted consumption of tree nuts should be advised for cashew allergic subjects.

Sa1758 In Vitro Adalimumab Exposure Decreases RORγT Expressing and RORγT/FoxP3 Double Expressing T-Lymphocytes From Peripheral Blood of Crohn's Disease Patients: A Study of Th17 Plasticity in Response to Anti-TNF

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.

Sa1757 Targeting Innate Immune Receptors to Treat Inflammatory Bowel Disease: Preclinical Activity of IMO-9200, an Antagonist of TLRS 7, 8, and 9 in Mouse Models of Colitis

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.

Sa1756 The Renin-Angiotensin System Promotes Colitis Independently of Blood Pressure

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.

Jug r 2–reactive CD4+ T cells have a dominant immune role in walnut allergy

BackgroundAllergic reactions to walnut can be life threatening. While IgE epitopes of walnut have been studied, CD4+ T-cell specific epitopes for walnut remain uncharacterized. Particularly, the relationship of both phenotype and frequency of walnut specific T-cells to the disease have not been examined.ObjectivesWe sought to provide a thorough phenotypic analysis for walnut reactive T-cells in allergic and non-allergic subjects. Particularly, the relationship of phenotypes and frequencies of walnut specific T-cells with the disease.MethodsCD154 up-regulation assay was used to examine CD4+ T-cell reactivity towards walnut allergens.Jug r 1, Jug r 2 and Jug r 3. Tetramer-Guided epitope mapping approach was utilized to identify HLA-restricted CD4+ T-cells epitopes in Jug r 2. Direct ex vivo staining with peptide-major histocompatibility complex class II (pMHC-II) tetramers enabled the comparison of frequency and phenotype of Jug r 2-specific CD4+ T-cells between allergic and non-allergic subjects. Jug r 2-specific T-cell-clones were also generated and mRNA transcription factor levels were assessed by RT qPCR. Intracellular cytokine staining (ICS) assays were performed for further phenotypical analyses.ResultsJug r 2 was identified as the major allergen that elicited CD4+ T-cell responses. Multiple Jug r 2 T-cell epitopes were identified. The majority of these T-cells in allergic subjects have a CCR4+ TCM (central memory) phenotype. A subset of these T-cells express CCR4+CCR6+ irrespectively of the asthmatic status of the allergic subjects. ICS confirmed these TH2, TH2/TH17 and TH17-like heterogenic profiles. Jug r 2-specific T-cell-clones from allergic subjects mainly expressed GATA3; nonetheless, a portion of T-cell clones expressed either GATA3 and RORC, or RORC, confirming the presence of TH2, TH2/TH17 and TH17 cells.ConclusionsJug r 2 specific responses dominate walnut T-cell responses in subjects with walnut allergy. Jug r 2 central memory CD4+ cells and terminal effector T-cells were detected in peripheral blood with the central memory phenotype as the most prevalent phenotype. In addition to conventional TH2-cells, TH2/TH17 and TH17 cells were also detected in non-asthmatic and asthmatic subjects with walnut allergy. Understanding this T-cell heterogeneity may render better understanding of the disease manifestation.

Engineered FVIII-Specific Human T-Effector Cells: Can the T-Cell Receptor Modulate CD4+ T-Helper Phenotypes or Is It Just a “Switch”?

Engineered T cells are a vital component in the armamentarium of cellular therapies. In this presentation, we examine how human CD4+ T cells, genetically engineered to express a T-cell receptor (TCR) specific for a C2 domain epitope of the coagulation protein cofactor FVIII, can be skewed or polarized to different T-helper subsets. Two TCRs were cloned from Th2 and Th17/Th1 phenotyped CD4+ T cells isolated via a tetramer guided epitope mapping (TGEM) technique from a hemophilia A subject after clinical diagnosis of an inhibitor (neutralizing antibody) to FVIII given as replacement therapy. The two TCRs were cloned using a 5’ RACE with semi-nested PCR and transduced via a retroviral vector into healthy non-hemophilia A human donor CD4+ T cells. Based on proliferation and HLA class II tetramer staining data, engineered CD4+ T cells expressing the different cloned TCRs exhibited different avidities for the same C2 peptide (containing the epitope) over a dose titration curve, despite similar levels of TCR expression on the CD4 T-cell surface. IFN-γ, TNF-α, IL-6, and IL-10 cytokine production levels following stimulation with C2 peptide and DR1 antigen presenting cells, as measured by cytokine bead analysis, were significantly greater for the higher avidity TCR, which was cloned from a “Th2” phenotyped CD4+ T-cell clone. Interestingly, neither the engineered CD4+ T cells expressing the Th2 TCR nor the cells expressing the Th17/Th1 TCR produced cytokines characteristic of their respective original parental clones. Rather, they reflected the cytokine profiles of the donor populations used for transduction. These preliminary data led us to investigate how the different avidities of the two cloned TCRs can modulate the T-helper subset skewing/differentiation potential of engineered CD4+T cells. We hypothesized that the TCR is merely a switch that can activate or direct engineered CD4+ T cells to an antigen-specific response that would be skewed to the T-helper phenotypes of the cells prior to TCR transduction. We further hypothesized that this response could be modulated after TCR transduction according to the apparent tetramer avidity of the engineered cells. We successfully skewed the engineered human T-helper cells to Th1, Th2 and Th17 lineages, based on T-helper signature cytokine expression and the transcription factors T-bet, Gata3 and RORγt. Moreover, we observed that TCR transduction into naïve human CD4+ T cells did not itself affect the T-helper subset skewing of the cells. Preliminary experiments showed a trend toward Th2 skewing for the high avidity Th2 CD4+ T cells having an engineered TCR when they were cultured under either Th1 or Th2 polarizing conditions and stimulated with the C2 peptide, compared to the phenotypes obtained following stimulation of polyclonal CD4 T cells with anti-CD3. These studies will improve our designing of engineered TCRs for CD4+T-cell therapy, especially when concerns of T-helper effector function and plasticity are important to clinical outcomes.Supported by NIH RO1-HL061883 (DWS), funding from Bayer and CSL Behring (KPP) and intramural support from NIAID (EMS). We thank Dr. Arthur Thompson (Puget Sound Blood Center) for enrolling patients and we thank all blood donors. DisclosuresNo relevant conflicts of interest to declare.

Peptide-induced immune regulation by a promiscuous and immunodominant CD4T-cell epitope of Timothy grass pollen: a role of Cbl-b and Itch in regulation

BackgroundT-cell targeted peptide epitope tolerogens from grass pollen allergens may be useful in treating seasonal allergic rhinitis, but there is urgent need for optimisation of approaches from improved understanding of...

Design of less immunogenic therapeutic factor VIII proteins (P3169)

Abstract Hemophilia A patients lack functional factor VIII (FVIII), which is essential for normal blood clotting. Approximately 1 in 4 patients receiving replacement therapy by infusions of FVIII develop neutralizing antibodies that can cause serious bleeds. Tetramer-guided epitope mapping (TGEM) experiments identified an HLA-DRB1*01:01-restricted T-cell epitope in 2 subjects with missense substitution A2201P. The same epitope was identified using samples from a subject with severe hemophilia. T-cell clones were isolated by single-cell sorting and expansion of tetramer-positive CD4 T cells. Peptide-MHC binding assays using recombinant DR0101 and FVIII peptides with systematic arginine substitutions identified anchor residues F2196, M2199, A2201 and S2204. Recombinant FVIII-C2 domain with substitution F2196A abrogated proliferation of 4 clones. Six FVIII proteins with rational substitutions designed to interfere with epitope presentation (F2196A, F2196K, F2196L, M2199A, M2199W and M2199R) were produced in a BHK expression system. All had pro-coagulant activities, measured by both clotting and chromogenic assays, similar to that of wild-type FVIII. Their immunogenicity is now being evaluated. These experiments provide proof of principle for the design of less immunogenic, functional FVIII proteins targeted to specific subsets of patients.

Comment on “Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire”

We read with interest the article by Kwok et al. ([1][1]), which reported the tetramer-guided epitope mapping of protective Ag (PA)-specific CD4+ T cells from both anthrax-vaccine adsorbed vaccinees and naive individuals. We have evidence that the three putative epitopes reported by the authors

Ara h 1–reactive T cells in individuals with peanut allergy

Effective immunotherapy for peanut allergy is hampered by a lack of understanding of peanut-reactive CD4(+) T cells. To identify, characterize, and track Ara h 1-reactive cells in subjects with peanut allergy by using Ara h 1-specific class II tetramers. Tetramer-guided epitope mapping was used to identify the antigenic peptides within the peanut allergen Ara h1. Subsequently, HLA class II/Ara h 1-specific tetramers were used to determine the frequency and phenotype of Ara h 1-reactive T cells in subjects with peanut allergy. Cytokine profiles of Ara h 1-reactive T cells were also determined. Multiple Ara h 1 epitopes with defined HLA restriction were identified. Ara h 1-specific CD4(+) T cells were detected in all of the subjects with peanut allergy tested. Ara h 1-reactive T cells in subjects with allergy expressed CCR4 but did not express CRTH2. The percentage of Ara h1-reactive cells that expressed the β7 integrin was low compared with total CD4(+) T cells. Ara h 1- reactive cells that secreted IFN-γ, IL-4, IL-5, IL-10, and IL-17 were detected. In individuals with peanut allergy, Ara h 1-reactive T cells occurred at moderate frequencies, were predominantly CCR4(+) memory cells, and produced IL-4. Class II tetramers can be readily used to detect Ara h 1-reactive T cells in the peripheral blood of subjects with peanut allergy without in vitro expansion and would be effective for tracking peanut-reactive CD4(+) T cells during immunotherapy.

Reassessing the role of HLA‐DRB3 T‐cell responses: Evidence for significant expression and complementary antigen presentation

In humans, several HLA-DRB loci (DRB1/3/4/5) encode diverse beta-chains that pair with alpha-chains to form DR molecules on the surface of APC. While DRB1 and DRB5 have been extensively studied, the role of DRB3/4 products of DR52/DR53 haplotypes has been largely neglected. To clarify the relative expression of DRB3, we quantified DRB3 mRNA levels in comparison with DRB1 mRNA from the same haplotype in both B cells and monocytes, observing quantitatively significant DRB3 synthesis. In CD19+ cells, DRB1*03/11/13 was 3.5-fold more abundant than DRB3, but in CD14+ this difference was only two-fold. Monocytes also had lower overall levels of DR mRNA compared with B cells, which was confirmed by cell surface staining of DRB1 and DRB3. To evaluate the functional role of DRB3, tetramer-guided epitope mapping was used to detect T cells against tetanus toxin and several influenza antigens presented by DRB3*0101/0202 or DRB1*03/11/13. None of the epitopes discovered were shared among any of the DR molecules. Quantitative assessment of DRB3-tetanus toxin specific T cells revealed that they are present at similar frequencies as those observed for DRB1. These results suggest that DRB3 plays a significant role in antigen presentation with different epitopic preferences to DRB1. Therefore, DRB3, like DRB5, serves to extend and complement the peptide repertoire of DRB1 in antigen presentation.

H5N1 strain-specific Hemagglutinin CD4+ T cell epitopes restricted by HLA DR4

CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain-specific HLA class II restricted epitopes is essential for monitoring cellular strain-specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA441–460 and H5HA57–76 were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA57–76 epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA57–76 response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA57–76 immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.

Searching H5N1 Strain-Specific Hemagglutinin Epitope Restricted by HLA DR4 (130.3)

Abstract CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain specific HLA class II restricted epitopes is essential for monitoring cellular strain specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA441-460 and H5HA57-76 were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA57-76 epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA57-76 response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA57-76 immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.

Searching immunodominant epitopes prior to epidemic: HLA class II-restricted SARS-CoV spike protein epitopes in unexposed individuals

Identification of dominant T cell epitopes within newly emerging and re-emerging infectious organisms is valuable in understanding pathogenic immune responses and potential vaccine designs. However, difficulties in obtaining samples from patients or convalescent subjects have hampered research in this direction. We demonstrated a strategy, tetramer-guided epitope mapping, that specific CD4+ T cell epitopes can be identified by using PBMC from subjects that have not been exposed to the infectious organism. Sixteen HLA-DR0401- and 14 HLA-DR0701-restricted epitopes within spike protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) were identified. Among these, spike protein residues 159–171, 166–178, 449–461 and 1083–1097 were identified to contain naturally processed immunodominant epitopes based on strong in vitro T cell responses of PBMC (as assayed by tetramer staining) to intact spike protein stimulation. These immunodominant epitopes were confirmed in vivo in HLA-DR0401 transgenic mice by immunizing with spike protein. Furthermore, the epitope-specific T cells from naive donors secreted IFN-γ and IL-13 upon re-stimulation with corresponding tetramers. Our study demonstrates a strategy to determine potential immunodominant epitopes for emerging infectious pathogens prior to their epidemic circulation.

Identification of Specific Residues in a Human FVIII T-Cell Epitope That Are Essential for An HLA-DRA-DRB1*0101-Restricted T-Cell Response Associated with Inhibitor Development.

The development of antibodies that interfere with the pro-coagulant activity of factor VIII (FVIII), clinically referred to as “inhibitors”, can complicate the treatment of individuals with hemophilia A, and inhibitors are associated with significantly higher morbidity and mortality. The production of both allo- and auto-immune inhibitors depends on the involvement of helper T cells. In order to better understand T-cell signaling events, it is necessary to identify and characterize the relevant T-cell epitopes in FVIII, i.e. the linear amino acid sequences that bind to MHC Class II (HLA-DR) molecules on the surface of antigen-presenting cells (APCs). These peptide-Class II complexes are recognized in turn by T-cell receptors, and sustained interaction between T cells and APCs through these complexes results in T-cell activation. We previously identified an HLA-DRA-DRB1*0101 restricted T-cell epitope recognized by CD4+ T cells from a mild hemophilia A subject with missense mutation A2201P in the FVIII C2 domain. This individual developed a high-titer inhibitor after receiving FVIII infusions for hemostatic support during surgery (James et al., J. Thrombos. Haemostas. 5:2399–2407, 2007). The epitope was identified using tetramer-guided epitope mapping, in which biotinylated DR0101 protein was incubated with synthetic 20-mer peptides having overlapping sequences spanning the FVIII C2 domain. The DR0101 proteins were converted to tetramers by addition of PE-labeled streptavidin, and T cells with receptors that recognized particular peptide-loaded tetramers were detected by flow cytometry. Peptides with sequences corresponding to FVIII residues 2186–2205, 2187–2205 and 2194–2213 were shown to contain a DRB1*0101 restricted epitope, suggesting that the epitope resided within the overlapping sequence S2194–P2205. Competition binding assays indicated peptide FVIII2194-2213 bound to DR0101 with ~20-fold higher affinity than a similar peptide with the hemophilic substitution A2201P. In the present study, we precisely defined the minimal epitope within this region by determining the affinities of a series of modified peptides for monomeric DR0101 protein. Truncated peptides were used to identify the minimal epitope, and their affinities as well as those of of peptides with single arginine substitutions indicated putative “anchor” residues that fit into pockets within the DR0101 peptide-binding groove. Briefly, DR0101 was incubated with biotinylated influenza virus haemagglutinin 306–318 (bHA) in the presence of several concentrations of a truncated and/or sequence-modified peptide. DR0101-bound peptide was separated from free peptide by immunoprecipitation of DR0101, and bHA peptide bound to DR0101 was detected using europium-labeled streptavidin. In this assay effective competition is thus indicated by a reduced fluorescent signal. N- and C-terminal truncated peptides corresponding to FVIII residues 2195-2206, 2196–2206, 2197–2206, 2194–2211, 2194–2207, 2194–2206, 2194–2205, 2194–2204 and 2194–2203 were tested, and the results indicated the minimal epitope consisted of S2194–P2205, with F2196 and S2204 serving as anchors at positions 1 and 9, respectively. The affinities of a series of peptides corresponding to residues 2194–2205, with a single residue at each position substituted to arginine, were also evaluated. Reduced binding affinities of several peptides indicated that the DR0101 anchor residues are F2196, M2199, A2201 and S2204. Our identification of anchor residues that contribute affinity necessary for productive DR0101-peptide complex formation indicates sites in FVIII that could be modified by amino acid substitutions to modulate DRB1*0101-restricted T-cell responses. The binding of these peptides to other prevalent DR molecules is currently being evaluated to indicate whether this epitope is immunodominant. Modification of immunodominant epitopes in next-generation recombinant FVIII proteins may eventually provide therapeutic benefits, especially in conjunction with clinical tolerance-inducing regimes, as reduced T-cell signaling could lead to lower antibody titres.

The Anthrax Vaccine Adsorbed Vaccine Generates Protective Antigen (PA)-Specific CD4 + T Cells with a Phenotype Distinct from That of Naïve PA T Cells

Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4(+) T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4(+) T cells with a CD45RA(-) phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4(+) T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA(+) and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4(+) T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4(+) T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4(+) cell responses and lineage development after vaccination.