Proinsulin-Reactive CD4 T Cells in the Islets of Type 1 Diabetes Organ Donors

Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for development of antigen-specific immunotherapies for type 1 diabetes (T1D). In this study, we sought to comprehensively evaluate reactivity to preproinsulin by CD4 T cells originally isolated from pancreatic islets of organ donors having T1D. We analyzed 187 T cell receptor (TCR) clonotypes expressed by CD4 T cells obtained from six T1D donors and determined their response to 99 truncated preproinsulin peptide pools, in the presence of autologous B cells. We identified 14 TCR clonotypes from four out of the six donors that responded to preproinsulin peptides. Epitopes were found across all of proinsulin (insulin B-chain, C-peptide, and A-chain) including four hot spot regions containing peptides commonly targeted by TCR clonotypes derived from multiple T1D donors. Of importance, these hot spots overlap with peptide regions to which CD4 T cell responses have previously been detected in the peripheral blood of T1D patients. The 14 TCR clonotypes recognized proinsulin peptides presented by various HLA class II molecules, but there was a trend for dominant restriction with HLA-DQ, especially T1D risk alleles DQ8, DQ2, and DQ8-trans. The characteristics of the tri-molecular complex including proinsulin peptide, HLA-DQ molecule, and TCR derived from CD4 T cells in islets, provides an essential basis for developing antigen-specific biomarkers as well as immunotherapies.

Download Full-text

HLA class II mediates type 1 diabetes risk by anti-insulin repertoire selection

10.1101/2021.09.06.458974 ◽

2021 ◽

Author(s):

Arcadio Rubio Garcia ◽

Athina Paterou ◽

Mercede Lee ◽

Hubert Slawinski ◽

Ricardo Ferreira ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Cd4 T Cells ◽

Pancreatic Beta Cells ◽

Class Ii ◽

Hla Class Ii ◽

Amino Acid Sequences ◽

Cell Repertoire

Type 1 diabetes (T1D) is a common autoimmune disorder characterized by the destruction of insulin-secreting pancreatic beta cells, in which polymorphism of the human leukocyte antigen (HLA) class II region is the major genetic risk factor. However, how variation in class II molecules alters T1D risk remains a longstanding question. Here we show how T1D risk due to HLA class II haplotype combinations correlates with the frequency of negatively charged sequences in the CDR3β region of CD4+ T cell receptor (TCR) repertoires purified from peripheral blood. These sequences are known to be common in receptors that bind insulin B:9-23, the primary autoantigen in T1D. We also show the same effect in circulating activated CD4+ T cells from newly-diagnosed T1D cases, and in islet-infiltrating T cells from patients with active T1D. Furthermore, we demonstrate that the proportion of insulin-reactive CD4+ T cells present in islets is predicted by the frequency of these negatively charged CDR3β amino acid sequences. Our results suggest diagnostic uses of T cell repertoire profiling in early detection of insulin autoimmunity, and inform ongoing efforts to improve tolerance induction to insulin and prevention of T1D.

Download Full-text

Targeting transcriptional coregulator OCA-B/Pou2af1 blocks activated autoreactive T cells in the pancreas and type-1 diabetes

10.1101/2020.02.06.937839 ◽

2020 ◽

Author(s):

Heejoo Kim ◽

Jelena Perovanovic ◽

Arvind Shakya ◽

Zuolian Shen ◽

Cody N. German ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Target Genes ◽

Autoimmune Diabetes ◽

Nod Mice ◽

Cell Receptor ◽

Glucose Levels ◽

Transcriptional Coregulator

AbstractThe transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present, but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice, but diminished in monoclonal models specific to artificial or neoantigens. Rationally-designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels, and reduced T cell infiltration and proinflammatory cytokine expression in newly-diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.~40-word summary statement for the online JEM table of contents and alertsKim and colleagues show that OCA-B in T cells is essential for the generation of type-1 diabetes. OCA-B loss leaves the pancreatic lymph nodes largely undisturbed, but associates autoreactive CD4+ T cells in the pancreas with anergy while deleting potentially autoreactive CD8+ T cells.SummaryKim et al. show that loss or inhibition of OCA-B in T cells protects mice from type-1 diabetes.

Download Full-text

Regulatory vs. inflammatory cytokine T-cell responses to mutated insulin peptides in healthy and type 1 diabetic subjects

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502967112 ◽

2015 ◽

Vol 112 (14) ◽

pp. 4429-4434 ◽

Cited By ~ 38

Author(s):

Maki Nakayama ◽

Kristen McDaniel ◽

Lisa Fitzgerald-Miller ◽

Carol Kiekhaefer ◽

Janet K. Snell-Bergeon ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Risk Allele ◽

T Cell Responses ◽

Cell Responses ◽

Control Subjects ◽

Risk Alleles ◽

Hla Dq

Certain class II MHC (MHCII) alleles in mice and humans confer risk for or protection from type 1 diabetes (T1D). Insulin is a major autoantigen in T1D, but how its peptides are presented to CD4 T cells by MHCII risk alleles has been controversial. In the mouse model of T1D, CD4 T cells respond to insulin B-chain peptide (B:9–23) mimotopes engineered to bind the mouse MHCII molecule, IAg7, in an unfavorable position or register. Because of the similarities between IAg7 and human HLA-DQ T1D risk alleles, we examined control and T1D subjects with these risk alleles for CD4 T-cell responses to the same natural B:9–23 peptide and mimotopes. A high proportion of new-onset T1D subjects mounted an inflammatory IFN-γ response much more frequently to one of the mimotope peptides than to the natural peptide. Surprisingly, the control subjects bearing an HLA-DQ risk allele also did. However, these control subjects, especially those with only one HLA-DQ risk allele, very frequently made an IL-10 response, a cytokine associated with regulatory T cells. T1D subjects with established disease also responded to the mimotope rather than the natural B:9–23 peptide in proliferation assays and the proliferating cells were highly enriched in certain T-cell receptor sequences. Our results suggest that the risk of T1D may be related to how an HLA-DQ genotype determines the balance of T-cell inflammatory vs. regulatory responses to insulin, having important implications for the use and monitoring of insulin-specific therapies to prevent diabetes onset.

Download Full-text

Differentiating MHC-dependent and -independent mechanisms of lymph node stromal cell regulation of proinsulin-specific CD8+ T-cells in type 1 diabetes

10.2337/figshare.13146209.v1 ◽

2020 ◽

Author(s):

Ada Admin ◽

Terri C. Thayer ◽

Joanne Davies ◽

James A. Pearson ◽

Stephanie J. Hanna ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Lymph Node ◽

T Cell ◽

Cell Receptor ◽

Peripheral Tolerance ◽

Beta Cell Destruction ◽

Nonobese Diabetic ◽

Autoreactive Cells

Lymph node stromal cells (LNSC) are essential for providing and maintaining peripheral self-tolerance of potentially autoreactive cells. In type 1 diabetes, proinsulin-specific CD8+T-cells, escaping central and peripheral tolerance, contribute to beta-cell destruction. Using G9Cα-/-CD8+T-cells specific for proinsulin, we studied the mechanisms by which LNSC regulate low-avidity autoreactive cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes. Whereas MHC-matched NOD-LNSC significantly reduced G9Cα-/-CD8+T-cell cytotoxicity and DC-induced proliferation, they failed to sufficiently regulate T-cells stimulated by anti-CD3/CD28. In contrast, non-MHC matched, control C57BL/6 mouse LNSC suppressed T-cell receptor engagement by anti-CD3/CD28 via MHC-independent mechanisms. This C57BL/6-LNSC suppression was maintained even after removal of the LNSC, demonstrating a direct effect of LNSC on T-cells, modifying antigen sensitivity and effector function. Thus, our results suggest that a loss of NOD-LNSC MHC-independent suppressive mechanisms may contribute to diabetes development.

Download Full-text

Using the T Cell Receptor as a Biomarker in Type 1 Diabetes

Frontiers in Immunology ◽

10.3389/fimmu.2021.777788 ◽

2021 ◽

Vol 12 ◽

Author(s):

Maki Nakayama ◽

Aaron W. Michels

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Current Knowledge ◽

Autoimmune Diabetes ◽

Cell Activity ◽

Cell Receptor ◽

Antigen Specificity ◽

Unique Markers

T cell receptors (TCRs) are unique markers that define antigen specificity for a given T cell. With the evolution of sequencing and computational analysis technologies, TCRs are now prime candidates for the development of next-generation non-cell based T cell biomarkers, which provide a surrogate measure to assess the presence of antigen-specific T cells. Type 1 diabetes (T1D), the immune-mediated form of diabetes, is a prototypical organ specific autoimmune disease in which T cells play a pivotal role in targeting pancreatic insulin-producing beta cells. While the disease is now predictable by measuring autoantibodies in the peripheral blood directed to beta cell proteins, there is an urgent need to develop T cell markers that recapitulate T cell activity in the pancreas and can be a measure of disease activity. This review focuses on the potential and challenges of developing TCR biomarkers for T1D. We summarize current knowledge about TCR repertoires and clonotypes specific for T1D and discuss challenges that are unique for autoimmune diabetes. Ultimately, the integration of large TCR datasets produced from individuals with and without T1D along with computational ‘big data’ analysis will facilitate the development of TCRs as potentially powerful biomarkers in the development of T1D.

Download Full-text

Alteration at a Single Amino Acid Residue in the T Cell Receptor α Chain Complementarity Determining Region 2 Changes the Differentiation of Naive Cd4 T Cells in Response to Antigen from T Helper Cell Type 1 (Th1) to Th2

Journal of Experimental Medicine ◽

10.1084/jem.191.12.2065 ◽

2000 ◽

Vol 191 (12) ◽

pp. 2065-2074 ◽

Cited By ~ 45

Author(s):

J. Magarian Blander ◽

Derek B. Sant'Angelo ◽

Kim Bottomly ◽

Charles A. Janeway

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cell Receptor ◽

T Helper Cell ◽

Cell Type ◽

T Helper ◽

Helper Cell Type ◽

Reference Peptide

To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR α chain of the D10.G4.1 (D10) TCR and bred them to D10 β chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by ∼100-fold. A mutation in the reference peptide CA134–146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex–bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.

Download Full-text

How C-terminal additions to insulin B-chain fragments create superagonists for T cells in mouse and human type 1 diabetes

Science Immunology ◽

10.1126/sciimmunol.aav7517 ◽

2019 ◽

Vol 4 (34) ◽

pp. eaav7517 ◽

Cited By ~ 8

Author(s):

Yang Wang ◽

Tomasz Sosinowski ◽

Andrey Novikov ◽

Frances Crawford ◽

Janice White ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Mhc Class Ii ◽

Cell Receptor ◽

Proper Position ◽

Peptide Epitopes ◽

Human Type 1 Diabetes

In type 1 diabetes (T1D), proinsulin is a major autoantigen and the insulin B:9-23 peptide contains epitopes for CD4+ T cells in both mice and humans. This peptide requires carboxyl-terminal mutations for uniform binding in the proper position within the mouse IAg7 or human DQ8 major histocompatibility complex (MHC) class II (MHCII) peptide grooves and for strong CD4+ T cell stimulation. Here, we present crystal structures showing how these mutations control CD4+ T cell receptor (TCR) binding to these MHCII-peptide complexes. Our data reveal stricking similarities between mouse and human CD4+ TCRs in their interactions with these ligands. We also show how fusions between fragments of B:9-23 and of proinsulin C-peptide create chimeric peptides with activities as strong or stronger than the mutated insulin peptides. We propose transpeptidation in the lysosome as a mechanism that could accomplish these fusions in vivo, similar to the creation of fused peptide epitopes for MHCI presentation shown to occur by transpeptidation in the proteasome. Were this mechanism limited to the pancreas and absent in the thymus, it could provide an explanation for how diabetogenic T cells escape negative selection during development but find their modified target antigens in the pancreas to cause T1D.

Download Full-text

HLA-DQA1 and HLA-DQB1 Alleles, Conferring Susceptibility to Celiac Disease and Type 1 Diabetes, are More Expressed Than Non-Predisposing Alleles and are Coordinately Regulated

Cells ◽

10.3390/cells8070751 ◽

2019 ◽

Vol 8 (7) ◽

pp. 751 ◽

Cited By ~ 11

Author(s):

Farina ◽

Picascia ◽

Pisapia ◽

Barba ◽

Vitale ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Celiac Disease ◽

Cd4 T Cells ◽

High Expression ◽

Transcriptional Level ◽

Risk Alleles ◽

Hla Dq ◽

Hla Dqa1

HLA DQA1*05 and DQB1*02 alleles encoding the DQ2.5 molecule and HLA DQA1*03 and DQB1*03 alleles encoding DQ8 molecules are strongly associated with celiac disease (CD) and type 1 diabetes (T1D), two common autoimmune diseases (AD). We previously demonstrated that DQ2.5 genes showed a higher expression with respect to non-CD associated alleles in heterozygous DQ2.5 positive (HLA DR1/DR3) antigen presenting cells (APC) of CD patients. This differential expression affected the level of the encoded DQ2.5 molecules on the APC surface and established the strength of gluten-specific CD4+ T cells response. Here, we expanded the expression analysis of risk alleles in patients affected by T1D or by T1D and CD comorbidity. In agreement with previous findings, we found that DQ2.5 and DQ8 risk alleles are more expressed than non-associated alleles also in T1D patients and favor the self-antigen presentation. To investigate the mechanism causing the high expression of risk alleles, we focused on HLA DQA1*05 and DQB1*02 alleles and, by ectopic expression of a single mRNA, we modified the quantitative equilibrium among the two transcripts. After transfection of DR7/DR14 B-LCL with HLA-DQA1*05 cDNA, we observed an overexpression of the endogenous DQB1*02 allele. The DQ2.5 heterodimer synthesized was functional and able to present gluten antigens to cognate CD4+ T cells. Our results indicated that the high expression of alpha and beta transcripts, encoding for the DQ2.5 heterodimeric molecules, was strictly coordinated by a mechanism acting at a transcriptional level. These findings suggested that, in addition to the predisposing HLA-DQ genotype, also the expression of risk alleles contributed to the establishment of autoimmunity.

Download Full-text

Differentiating MHC-dependent and -independent mechanisms of lymph node stromal cell regulation of proinsulin-specific CD8+ T-cells in type 1 diabetes

10.2337/figshare.13146209 ◽

2020 ◽

Author(s):

Ada Admin ◽

Terri C. Thayer ◽

Joanne Davies ◽

James A. Pearson ◽

Stephanie J. Hanna ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Lymph Node ◽

T Cell ◽

Cell Receptor ◽

Peripheral Tolerance ◽

Beta Cell Destruction ◽

Nonobese Diabetic ◽

Autoreactive Cells

Download Full-text

Proinsulin-specific T regulatory cells may control immune responses in type 1 diabetes: implications for adoptive therapy

BMJ Open Diabetes Research & Care ◽

10.1136/bmjdrc-2019-000873 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000873 ◽

Cited By ~ 1

Author(s):

Mateusz Gliwiński ◽

Dorota Iwaszkiewicz-Grześ ◽

Anna Wołoszyn-Durkiewicz ◽

Monika Tarnowska ◽

Magdalena Żalińska ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Immune Responses ◽

T Regulatory Cells ◽

Cell Receptor ◽

Regulatory Cells ◽

Antigen Specificity ◽

Adoptive Therapy

ObjectiveHere we looked for possible mechanisms regulating the progression of type 1 diabetes mellitus (T1DM). In this disease, autoaggressive T cells (T conventional cells, Tconvs) not properly controlled by T regulatory cells (Tregs) destroy pancreatic islets.Research design and methodsWe compared the T-cell compartment of patients with newly diagnosed T1DM (NDT1DM) with long-duration T1DM (LDT1DM) ones. The third group consisted of patients with LDT1DM treated previously with polyclonal Tregs (LDT1DM with Tregs). We have also looked if the differences might be dependent on the antigen specificity of Tregs expanded for clinical use and autologous sentinel Tconvs.ResultsPatients with LDT1DM were characterized by T-cell immunosenescence-like changes and expansion of similar vβ/T-cell receptor (TCR) clones in Tconvs and Tregs. The treatment with Tregs was associated with some inhibition of these effects. Patients with LDT1DM possessed an increased percentage of various proinsulin-specific T cells but not GAD65-specific ones. The percentages of all antigen-specific subsets were higher in the expansion cultures than in the peripheral blood. The proliferation was more intense in proinsulin-specific Tconvs than in specific Tregs but the levels of some proinsulin-specific Tregs were exceptionally high at baseline and remained higher in the expanded clinical product than the levels of respective Tconvs in sentinel cultures.ConclusionsT1DM is associated with immunosenescence-like changes and reduced diversity of T-cell clones. Preferential expansion of the same TCR families in both Tconvs and Tregs suggests a common trigger/autoantigen responsible. Interestingly, the therapy with polyclonal Tregs was associated with some inhibition of these effects. Proinsulin-specific Tregs appeared to be dominant in the immune responses in patients with T1DM and probably associated with better control over respective autoimmune Tconvs.Trial registration numberEudraCT 2014-004319-35.

Download Full-text