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. 2017 Mar;66(3):722-734.
doi: 10.2337/db16-1025. Epub 2016 Dec 5.

Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes

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Free PMC article

Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes

Aaron W Michels et al. Diabetes. .
Free PMC article

Abstract

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Figures

Figure 1
Figure 1
Pancreas histology and T-cell infiltration in pancreatic islets of the three studied organ donors with type 1 diabetes. Immunohistochemical staining of serial paraffin sections from the pancreata of the three organ donors. Donors 6323 (A and B), 6342 (D and E), and 69 (G and H) were stained for Ki67 (brown) and insulin (red) (A, D, and G) or CD3 (brown) and glucagon (red) (B, E, and H). All donors have islet-infiltrating CD3-positive cells, whereas donors 6323 and 6342 have residual insulin staining in islets. Flow-cytometric analysis of CD4 and CD8 T cells infiltrating the pancreatic islets of donors 6323 (C), 6342 (F), and 69 (I).
Figure 2
Figure 2
TCR sequencing of T cells in the pancreatic islets of organ donors with type 1 diabetes. A: Strategy to test the antigen specificity of T cells infiltrating pancreatic islets. CD4 and CD8 T cells were isolated from islets by single-cell sorting, followed by amplification and sequencing of individual TCRs. “Artificial” T cells, termed T-cell transductants, were generated using retroviral vectors encoding the detected TCR sequences of interest such that immortalized cells were made expressing only a single TCR. The TCR transductants were then tested for a functional response to candidate antigens, including overlapping peptides derived from preproinsulin. B: Diversity of TCR sequences of T cells infiltrating pancreatic islets in the organ donors with type 1 diabetes. Several to hundreds of TCR α and β paired sequences were determined from each individual donor. White represents sequences detected only once, gray twice, and black three or more times. CD4 TCR sequences exhibit more diversity, whereas CD8 repertories have more clonality than CD4 in all three patients. C: TCR sequences detected two or more times in separated islet preparations from a single patient. CD4 T cells for single-cell analysis were isolated from two separate islet preparations for donors 6323 and 6342. Of TCR sequences detected repeatedly, the numbers of those detected in each islet preparation are shown. The majority of repeatedly detected TCRs were found from multiple islet preparations in the same patient.
Figure 3
Figure 3
Screening of islet-infiltrating TCR transductants for response to preproinsulin and islet peptides. (A) Heatmap depicts antigen-stimulated T-cell transductants derived from receptor sequencing of CD4 T cells. Responses are shown for each individual T-cell transductant, measured by IL-2 secretion, to overlapping preproinsulin peptides (B) and all tested islet peptides (C) in the presence of a mixture of antigen-presenting cells expressing DQ2 or DQ8, DR3 or DR4, or DQ2-trans or DQ8-trans. Each row denotes an individual TCR transductant with 38 tested from donor 6323, 40 from donor 6342, and 7 from donor 69. The peptide number in the columns of the heatmap corresponds to those in panel C. Three T cells, named GSE.8E3, GSE.6H9, and GSE.20D11, responding to proinsulin peptides were identified.
Figure 4
Figure 4
Response to antigens and islets by insulin B:9–23–reactive T cells identified within pancreatic islets and peripheral blood. T-cell transductants GSE.8E3 (A), GSE.6H9 (B), and GSE.20D11 (C) were cultured with cognate peptides C:19–35 for GSE.8E3 (A) or insulin B:9–23 and its mimotope B:9–23 (B22E) for GSE.6H9 and GSE.20D11 (B and C) in the presence of murine B cell lines expressing DQ2, DQ8, DQ2-trans, or DQ8-trans to determine HLA restriction. D–G: T-cell transductants expressing proinsulin-reactive TCRs derived from the islets (GSE.8E3, GSE.6H9, GSE.20D11), those previously cloned from peripheral blood of other patients with type 1 diabetes (T1D3, T1D10, and Clone 5), or an α-gliadin–responsive TCR (489) as a control were tested for reactivity to proinsulin, islets, and insulin B:9–23 peptides. The T-cell transductants were cultured with insulin B:9–23 peptides (D), whole proinsulin in the presence of human monocyte-derived dendritic cells derived from a DQ8/DQ2 heterozygous individual (E), whole proinsulin in the presence of the K562 human myelogenous cells expressing DQ8 (for all other than GSE.8E3 TCRs) or DQ8-trans (for GSE.8E3) (F), whole proinsulin in the presence of DQ8-transgenic spleen cells (G), islets isolated from T cell–deficient NOD mice, and human organ donors without diabetes in the presence of DQ8-transgenic spleen cells (H). Levels of IL-2 secreted into the tissue culture supernatant were measured by a highly sensitive ELISA. Distinct differences in response to whole proinsulin and islets are noted between insulin B:9–23–reactive T cells based upon the originating tissue location. All data shown are representative of at least three independent experiments. *P < 0.01 by two-tailed unpaired t test. Ag, antigen.

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