Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic β-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
peptide, found in the normal human gut commensal
, 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
insB:9-23 peptide mimic or insB:9-23 peptide verified immune cross-reactivity. Colonization of female NOD mice with
accelerated the development of T1D, increasing macrophages, dendritic cells, and destructive CD8
T cells, while decreasing FoxP3
regulatory T cells. Western blot analysis identified
-reacting antibodies in sera of NOD mice colonized with
and human T1D patients. Furthermore, adoptive transfer of splenocytes from
-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
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.
Posttranslational modification (PTM) of self-proteins has been shown to elicit clinically relevant immune responses in rheumatoid arthritis and celiac disease. Accumulating evidence suggests that ...recognition of modified self-proteins may also be important in type 1 diabetes. Our objective was to identify posttranslationally modified GAD65 peptides, which are recognized by subjects with type 1 diabetes, and to assess their disease relevance. We show that citrullination and transglutamination of peptides can enhance their binding to DRB1*04:01, a diabetes-susceptible HLA allele. These and corresponding modifications to amino acids at T-cell contact positions modulated the recognition of multiple GAD65 peptides by self-reactive T cells. Using class II tetramers, we verified that memory T cells specific for these modified epitopes were detectable directly ex vivo in the peripheral blood of subjects with type 1 diabetes at significantly higher frequencies than healthy controls. Furthermore, T cells that recognize these modified epitopes were either less responsive or nonresponsive to their unmodified counterparts. Our findings suggest that PTM contributes to the progression of autoimmune diabetes by eliciting T-cell responses to new epitope specificities that are present primarily in the periphery, thereby circumventing tolerance mechanisms.
Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer ...cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal 3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.
Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA⁹⁷ in a weakly bound ...register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramere loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.
MHC tetramers are an essential tool for characterizing antigen-specific CD4+ T cells. However, their ex vivo analysis is limited by the large sample requirements. Here we demonstrate a combinatorial ...staining approach that allows simultaneous characterization of multiple specificities to address this challenge. As proof of principle, we analyse CD4+ T-cell responses to the seasonal influenza vaccine, establishing a frequency hierarchy and examining differences in memory and activation status, lineage commitment and cytokine expression. We also observe cross-reactivity between an established epitope and recent variant and provide a means for probing T-cell receptor cross-reactivity. Using cord blood samples, we correlate the adult frequency hierarchy with the naive precursor frequencies. Last, we use our combinatorial staining approach to demonstrate that rheumatoid arthritis patients on therapy can mount effective responses to influenza vaccination. Together, these results demonstrate the utility of combinatorial tetramer staining and suggest that this approach may have broad applicability in human health and disease.
T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. ...Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who achieved a complete response following adoptive transfer of tumor-infiltrating lymphocytes (TILs). Tumor exome sequencing surprisingly revealed fewer than 30 nonsynonymous somatic mutations, including oncogenic BRAFV600E. Analysis of the specificity of TILs identified rare CD4+ T cells specific for BRAFV600E and diverse CD8+ T cells reactive to nonmutated self-antigens. These specificities increased in blood after TIL transfer and persisted long-term, suggesting they contributed to the effective antitumor immune response. Gene transfer of the BRAFV600E-specific T cell receptor (TCR) conferred recognition of class II MHC-positive cells expressing the BRAF mutation. Therapy with TCR-engineered BRAFV600E-specific CD4+ T cells may have direct antitumor effects and augment CD8+ T cell responses to self- and/or mutated tumor antigens in patients with BRAF-mutated cancers.
Susceptibility to type 1 diabetes (T1D) is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02). Monitoring T1D-specific T cell responses to ...DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65) using DQ8 tetramers. We demonstrated that GAD65 121-140 and GAD65 250-266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65 121-140 and GAD65 250-266 carried a Th1-dominant phenotype, with some of the GAD65 121-140-specific T cell clones producing IL-17. GAD65 250-266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs) revealed that GAD65 250-266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65 121-140 and GAD65 250-266 epitopes and implicate their possible contribution to the progression of T1D.
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.
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.
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.
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.
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Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8
+
tumor-infiltrating lymphocytes (TILs). Here, we study CD4
+
TILs ...in human lung and colorectal cancers and observe that non-Treg CD4
+
TILs average more than 70% of total CD4
+
TILs in both cancer types. Leveraging high dimensional analyses including mass cytometry, we reveal that CD4
+
TILs are phenotypically heterogeneous, within each tumor and across patients. Consistently, we find different subsets of CD4
+
TILs showing characteristics of effectors, tissue resident memory (Trm) or exhausted cells (expressing PD-1, CTLA-4 and CD39). In both cancer types, the frequencies of CD39
-
non-Treg CD4
+
TILs strongly correlate with frequencies of CD39
-
CD8
+
TILs, which we and others have previously shown to be enriched for cells specific for cancer-unrelated antigens (bystanders). Ex-vivo, we demonstrate that CD39
-
CD4
+
TILs can be specific for cancer-unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4
+
TILs can also recognize cancer-unrelated antigens and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4
+
T cells.
Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, ...questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype.
To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8).
Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression.
These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function.
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