HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic ß-cells. One of the earliest aspects of this process is the development of autoantibodies and T cells directed at an epitope in the B-chain of insulin (insB:9-23). Analysis of microbial protein sequences with homology to the insB:9-23 sequence revealed 17 peptides showing >50% identity to insB:9-23. Of these 17 peptides, the hprt4-18 peptide, found in the normal human gut commensal Parabacteroides distasonis, activated both human T cell clones from T1D patients and T cell hybridomas from nonobese diabetic (NOD) mice specific to insB:9-23. Immunization of NOD mice with P. distasonis insB:9-23 peptide mimic or insB:9-23 peptide verified immune cross-reactivity. Colonization of female NOD mice with P. distasonis accelerated the development of T1D, increasing macrophages, dendritic cells, and destructive CD8+ T cells, while decreasing FoxP3+ regulatory T cells. Western blot analysis identified P. distasonis-reacting antibodies in sera of NOD mice colonized with P. distasonis and human T1D patients. Furthermore, adoptive transfer of splenocytes from P. distasonis-treated mice to NOD/SCID mice enhanced disease phenotype in the recipients. Finally, analysis of human children gut microbiome data from a longitudinal DIABIMMUNE study revealed that seroconversion rates (i.e., the proportion of individuals developing two or more autoantibodies) were consistently higher in children whose microbiome harbored sequences capable of producing the hprt4-18 peptide compared to individuals who did not harbor it. Taken together, these data demonstrate the potential role of a gut microbiota-derived insB:9-23-mimic peptide as a molecular trigger of T1D pathogenesis.

Correction: Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19.

[This corrects the article DOI: 10.1371/journal.ppat.1009842.].

Distinct trajectories distinguish antigen-specific T cells in peanut-allergic individuals undergoing oral immunotherapy.

BACKGROUND: Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions. OBJECTIVE: This study aimed to determine whether inherent qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT). METHODS: We first performed ex vivo T-cell profiling on peanut-reactive CD154+CD137+ T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4+ T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT. RESULTS: Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6-CRTH2+ and CCR6+CRTH2-). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of TH2A pTeff cells. PnOIT induced elimination of TH2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of TH2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation. CONCLUSION: Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies.

Cross-reactive and mono-reactive SARS-CoV-2 CD4+ T cells in prepandemic and COVID-19 convalescent individuals.

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.

Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19.

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0-79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein, and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein.

Immunological Interaction of HLA-DPB1 and Proteinase 3 in ANCA Vasculitis is Associated with Clinical Disease Activity.

BACKGROUND: PR3-ANCA vasculitis has a genetic association with HLA-DPB1. We explored immunologic and clinical features related to the interaction of HLA-DPB1*04:01 with a strongly binding PR3 peptide epitope (PR3225-239). METHODS: Patients with ANCA vasculitis with active disease and disease in remission were followed longitudinally. Peripheral blood mononuclear cells from patients and healthy controls with HLA-DPB1*04:01 were tested for HLA-DPB1*04:01 expression and interaction with a PR3 peptide identified via in silico and in vitro assays. Tetramers (HLA/peptide multimers) identified autoreactive T cells in vitro. RESULTS: The HLA-DPB1*04:01 genotype was associated with risk of relapse in PR3-ANCA (HR for relapse 2.06; 95% CI, 1.01 to 4.20) but not in myeloperoxidase (MPO)-ANCA or the combined cohort. In silico predictions of HLA and PR3 peptide interactions demonstrated strong affinity between ATRLFPDFFTRVALY (PR3225-239) and HLA-DPB1*04:01 that was confirmed by in vitro competitive binding studies. The interaction was tested in ex vivo flow cytometry studies of labeled peptide and HLA-DPB1*04:01-expressing cells. We demonstrated PR3225-239 specific autoreactive T cells using synthetic HLA multimers (tetramers). Patients in long-term remission off therapy had autoantigenic peptide and HLA interaction comparable to that of healthy volunteers. CONCLUSIONS: The risk allele HLA-DPB1*04:01 has been associated with PR3-ANCA, but its immunopathologic role was unclear. These studies demonstrate that HLA-DPB1*04:01 and PR3225-239 initiate an immune response. Autoreactive T cells specifically recognized PR3225-239 presented by HLA-DPB1*04:01. Although larger studies should validate these findings, the pathobiology may explain the observed increased risk of relapse in our cohort. Moreover, lack of HLA and autoantigen interaction observed during long-term remission signals immunologic nonresponsiveness.

Evaluating Responses to Gluten Challenge: A Randomized, Double-Blind, 2-Dose Gluten Challenge Trial.

BACKGROUND & AIMS: Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology. METHODS: In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence. RESULTS: All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose-response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot, gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2-restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or near significantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure. CONCLUSIONS: Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796.

Specific T cells targeting Staphylococcus aureus fibronectin-binding protein 1 induce a type 2/type 1 inflammatory response in sensitized atopic dermatitis patients.

BACKGROUND: Atopic dermatitis (AD) is one of the most common inflammatory skin diseases worldwide and Staphylococcus aureus colonization and secondary infections occur in the majority of AD patients. Allergic sensitizations against microbial antigens have been discussed as possible trigger factors of AD. Recently, we reported IgE sensitization against fibronectin-binding protein 1 (FBP1), an essential virulence component in S. aureus, in a subgroup of patients suffering from AD. To expand these findings by investigating delayed-type immune reactions, the objective of this study was to detect and phenotypically characterize FBP1-specific T cells as possible trigger factors in AD. METHODS: Immunodominant T-cell epitopes were mapped by proliferation testing of patient-derived FBP1-specific T-cell lines after stimulation with single 15mer peptides, which were derived from different functional domains of the FBP1 sequence. Major histocompatibility complex class II tetramers carrying immunodominant epitopes successfully stained T helper cells in 8 out of 8 HLA-matched, IgE-sensitized AD patients. RESULTS: Cytokine profiling of multimer-sorted cells revealed that predominantly the type 2 cytokines IL-13 and IL-4 were secreted by these cells. In contrast, IL-17, the marker cytokine for response to extracellular pathogens, was scarcely detectable. CONCLUSIONS: We demonstrate that FBP1 contains immunodominant peptides that induce a specific pro-inflammatory T helper cell response with increased Th2 levels that can drive an allergic inflammation in sensitized AD patients.

Epitope Selection for HLA-DQ2 Presentation: Implications for Celiac Disease and Viral Defense.

We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class II. In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2α+53G. The APC pairs were compared for their ability to stimulate human CD4+ T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhigh APC expressing the DQ2α+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo.

C-terminal modification of the insulin B:11-23 peptide creates superagonists in mouse and human type 1 diabetes.

A polymorphism at ß57 in some major histocompatibility complex class II (MHCII) alleles of rodents and humans is associated with a high risk for developing type 1 diabetes (T1D). However, a highly diabetogenic insulin B chain epitope within the B:9-23 peptide is presented poorly by these alleles to a variety of mouse and human CD4 T cells isolated from either nonobese diabetic (NOD) mice or humans with T1D. We have shown for both species that mutations at the C-terminal end of this epitope dramatically improve presentation to these T cells. Here we present the crystal structures of these mutated peptides bound to mouse IAg7 and human HLA-DQ8 that show how the mutations function to improve T-cell activation. In both peptide binding grooves, the mutation of B:22R to E in the peptide changes a highly unfavorable side chain for the p9 pocket to an optimal one that is dependent on the ß57 polymorphism, accounting for why these peptides bind much better to these MHCIIs. Furthermore, a second mutation of the adjacent B:21 (E to G) removes a side chain from the surface of the complex that is highly unfavorable for a subset of NOD mouse CD4 cells, thereby greatly enhancing their response to the complex. These results point out the similarities between the mouse and human responses to this B chain epitope in T1D and suggest there may be common posttranslational modifications at the C terminus of the peptide in vivo to create the pathogenic epitopes in both species.

Human TH1 and TH2 cells targeting rhinovirus and allergen coordinately promote allergic asthma.

BACKGROUND: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. OBJECTIVE: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. METHODS: Circulating virus-specific TH1 cells and allergen-specific TH2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. RESULTS: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1+ TH1 cells, but not allergen-specific TH2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral TH1 response in asthmatic subjects versus controls, with synchronized allergen-specific TH2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, TH2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-γ, TNF-α) correlated with both egress of circulating virus-specific TH1 cells and worse symptoms. CONCLUSIONS: Rhinovirus induces robust TH1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

Nasal allergen challenge and environmental exposure chamber challenge: A randomized trial comparing clinical and biological responses to cat allergen.

BACKGROUND: The direct-instillation nasal allergen challenge (NAC) and the environmental exposure chamber (EEC) are 2 methods of conducting controlled allergen provocations. The clinical and biological comparability of these methods has not been thoroughly investigated. OBJECTIVE: We sought to compare clinical and immunologic responses to cat allergen in NAC versus EEC. METHODS: Twenty-four participants were randomized to receive either NAC followed by a 2-day challenge in an EEC or a 2-day challenge in an EEC followed by NAC. Challenges were separated by 28-day washout periods. We measured total nasal symptom scores, peak nasal inspiratory flow, nasal (0-8 hours) and serum cytokines, serum antibodies, peripheral blood antigen-specific T lymphocytes, and gene expression in nasal scrapings. The primary outcome was the total nasal symptom score area under the curve for the first 3 hours after allergen exposure in NAC or after initiation of exposure in EEC. RESULTS: Both challenges increased IL-5 and IL-13 in nasal fluids and serum and resulted in altered nasal cell expression of gene modules related to mucosal biology and transcriptional regulation. Changes in gene modules, more so than cytokine measurements, showed significant associations with total nasal symptom score and peak nasal inspiratory flow. Overall, EEC exposure generated larger responses and more early terminations compared with NAC. Although the 2 challenges did not correlate in symptom magnitude or temporality, striking correlations were observed in cytokine levels. CONCLUSIONS: Although clinical outcomes of NAC and EEC were temporally different and nonequivalent in magnitude, immunologic responses were similar. Selection of a particular allergen challenge method should depend on considerations of study objectives and cost.

Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis.

BACKGROUND: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. RESULTS: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-ß, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. CONCLUSIONS: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

Restricted myeloperoxidase epitopes drive the adaptive immune response in MPO-ANCA vasculitis.

BACKGROUND: Treatment of autoimmune diseases has relied on broad immunosuppression. Knowledge of specific interactions between human leukocyte antigen (HLA), the autoantigen, and effector immune cells, provides the foundation for antigen-specific therapies. These studies investigated the role of HLA, specific myeloperoxidase (MPO) epitopes, CD4+ T cells, and ANCA specificity in shaping the immune response in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. METHODS: HLA sequence-based typing identified enriched alleles in our patient population (HLA-DPB1*04:01 and HLA-DRB4*01:01), while in silico and in vitro binding studies confirmed binding between HLA and specific MPO epitopes. Class II tetramers with MPO peptides were utilized to detect autoreactive CD4+ T cells. TCR sequencing was performed to determine the clonality of T cell populations. Longitudinal peptide ELISAs assessed the temporal nature of anti-MPO447-461 antibodies. Solvent accessibility combined with chemical modification determined the buried regions of MPO. RESULTS: We identified a restricted region of MPO that was recognized by both CD4+ T cells and ANCA. The autoreactive T cell population contained CD4+CD25intermediateCD45RO+ memory T cells and secreted IL-17A. T cell receptor (TCR) sequencing demonstrated that autoreactive CD4+ T cells had significantly less TCR diversity when compared to naïve and memory T cells, indicating clonal expansion. The anti-MPO447-461 autoantibody response was detectable at onset of disease in some patients and correlated with disease activity in others. This region of MPO that is targeted by both T cells and antibodies is not accessible to solvent or chemical modification, indicating these epitopes are buried. CONCLUSIONS: These observations reveal interactions between restricted MPO epitopes and the adaptive immune system within ANCA vasculitis that may inform new antigen-specific therapies in autoimmune disease while providing insight into immunopathogenesis.

DRB4*01:01 Has a Distinct Motif and Presents a Proinsulin Epitope That Is Recognized in Subjects with Type 1 Diabetes.

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets. To identify high-affinity peptides that are recognized in the context of DRB4, we used an HLA class II tetramer-based approach to identify epitopes within multiple viral Ags. We applied a similar approach to identify antigenic sequences within glutamic acid decarboxylase 65 and pre-proinsulin that are recognized in the context of DRB4. Seven sequences were immunogenic, eliciting high-affinity T cell responses in DRB4+ subjects. DRB1*04:01-restricted responses toward many of these peptides have been previously described, but responses to a novel pre-proinsulin 9-28 peptide were commonly observed in subjects with T1D. Furthermore, T cells that recognized this peptide in the context of DRB4 were present at significantly higher frequencies in patients with T1D than in healthy controls, implicating this as a disease-relevant specificity that may contribute to the breakdown of ß cell tolerance in genetically susceptible individuals. We then deduced a DRB4 motif and confirmed its key features through structural modeling. This modeling suggested that the core epitope within the pre-proinsulin 9-28 peptide has a somewhat unusual binding motif, with tryptophan in the fourth binding pocket of DRB4, perhaps influencing the availability of this complex for T cell selection.

Regulatory CD4+ T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

BACKGROUND: CD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules. METHODS: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined. RESULTS: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs. CONCLUSIONS: These findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.

Analysis of pancreatic beta cell specific CD4+ T cells reveals a predominance of proinsulin specific cells.

CD4+ T cell responses are thought to play a role in type 1 diabetes (T1D). However, detection and characterization of T cells that respond to beta cell epitopes in subjects with T1D has been limited by technical obstacles, including the inherently low frequencies in peripheral blood and variable responsiveness of individual subjects to single epitopes. We implemented a multicolor staining approach that allows direct ex vivo characterization of multiple CD4+ T cell specificities in a single sample. Here we demonstrate and apply that multicolor approach to directly measure the frequency and phenotype of beta cell specific CD4+ T cells in T1D patients and HLA matched controls. For this work we utilized five DR0401 restricted peptides from proinsulin, GAD65, IA-2, and IGRP, which were previously reported as disease relevant epitopes. Surprisingly, although responses to each of these peptides can be readily detected after in vitro expansion, our results indicated that only proinsulin specific T cells were consistently detectable at moderate frequencies in subjects with T1D. Characterization of beta cell specific CD4+ T cells revealed only modest differences between subjects with T1D and healthy controls. Subjects with T1D did have higher proportions of CD45RA negative epitope specific T cells than controls. In patients epitope specific T cells were often CXCR3 positive and a substantial proportion were CCR7 negative, suggesting a Th1-like effector phenotype. Finally, we demonstrated that our multicolor staining approach is compatible with class I multimer analysis, facilitating the characterization of self-reactive CD4+ and CD8+ T cells using a single sample.

Modulation of CRTh2 expression on allergen-specific T cells following peptide immunotherapy.

BACKGROUND: Allergen immunotherapy using synthetic peptide T-cell epitopes (Cat-PAD) from the major cat allergen Fel d 1 has been shown, in allergen exposure studies, to significantly reduce symptoms of allergic rhinoconjunctivitis in cat-allergic subjects. However, the immunological mechanisms underlying clinical benefit remain only partially understood. Since previous studies of whole allergen immunotherapy demonstrated a reduction in the frequency of allergen-specific (MHC II tetramer+ ) CD4+ T cells expressing the chemokine receptor CRTh2, we assessed the impact of Cat-PAD on the frequency and functional phenotype of Fel d 1-specific CD4+ T cells. METHODS: Using before and after treatment samples from subjects enrolled in a randomized, double-blind, placebo-controlled trial of Cat-PAD, we employed Fel d 1 MHC II tetramers and flow cytometry to analyze the expression of chemokine receptors CCR3, CCR4, CCR5, CXCR3, and CRTh2, together with markers of memory phenotype (CD27 and CCR7) on Fel d 1-specific CD4+ T cells. RESULTS: No statistically significant change in the frequency of Fel d 1-specific CD4+ T cells, nor in their expression of chemokine receptors or memory phenotype, was observed. However, a significant reduction in cell surface expression of CRTh2 was observed between the placebo and active groups (P = 0.047). CONCLUSIONS: Peptide immunotherapy with Cat-PAD does not significantly alter the frequency or phenotype of Fel d 1-CD4+ T cells, but may decrease their expression of CRTh2.