Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes

Abstract

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8⁺ T cells in situ, in islets of human leukocyte antigen (HLA)-A2⁺ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4⁺ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4⁺ and CD8⁺ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Figure 1

Schema of islet handling and ex vivo isolation and growth of T cells from islets. (a) Isolated islets were received and handpicked to increase purity. To capture the maximum number of islet-infiltrating T cells, an aliquot of 100 handpicked islets was dispersed with enzyme, stained for CD45, CD3, CD19, CD4, CD8 and Zombie Violet viability dye. All detectable CD45⁺CD3⁺CD4⁺ and CD45⁺CD3⁺CD8⁺ T cells were single-cell sorted by flow cytometry (Supplementary Table 1) and cultured for 1–3 rounds (4–6 weeks) with irradiated allogeneic feeders, PHA-P and IL-2, IL-7 and IL-15. *The frequency of CD4⁺ and CD8⁺ T cells detected by ex vivo flow cytometry from dispersed islets is shown in Supplementary Tables 1 and 2. Alternatively, aliquots of 100 handpicked islets were cultured on a gel matrix with soluble anti-CD3, anti-CD28, anti-Fas, anti-PD-1, IL-2, IL-4, IL-7, IL-15 and mifepristone. After 5–10 d of culture, cellular outgrowth from islets was recovered under a dissecting microscope and cultured as above with irradiated allogeneic feeders, PHA-P and IL-2, IL-7 and IL-15. Surface expression of CD4⁺ or CD8⁺ was determined or confirmed by flow cytometry. **Numbers of T cell lines and clones grown are shown in Supplementary Tables 1 and 2. (b,c) Representative flow cytometric analysis of dispersed islets from a donor with T1D (nPOD69) (b) and a donor without T1D (Is.7) (c). Not shown, forward scatter (FSC) versus side scatter (SSC) panels with cells stained with viability dye. Frequency of cell subsets is shown. In the FSC and SSC panels, the colored cells indicate the origin of the positively sorted cells shown in the subsequent panels (CD8, red; CD4, green; and CD4^-CD8^-, purple). (d) Outgrowth of T cells from an islet remnant from nPOD69, cultured as described on the top line of the schematic in a. Scale bar, 60 μm.

Figure 2

Detection of reactivity to known autoreactive targets of CD4⁺ and CD8⁺ T cell lines and clones sorted or directly grown from islets from donors with T1D. T cell lines and clones were tested for reactivity against known autoreactive peptide and protein targets presented by B cells (described below for each panel) as described in Online Methods. The sample source and designation of each line or clone are listed, along with the HLA restriction, if determined (in italics). (a) A CD4⁺ T cell line grown directly from an islet from donor T1D.6 recognized GAD_555–567 in the context of a bare lymphocyte syndrome (BLS) B cell expressing only HLA-DRA1*01:01 and HLA-DRB1*04:01. (b) A CD4⁺ T cell line grown directly from an islet from donor nPOD69 recognized proinsulin_76–90 in the context of a BLS B cell expressing only HLA-DRA1*01:01 and HLA-DRB1*04:01. (c) A CD4⁺ T cell clone sorted from islets from donor nPOD6342 recognized GAD_274–286 by the secretion of IFN-γ in the context of Priess B cells. (d) A CD4⁺ T cell clone sorted from the islets from donor T1D.7 recognized GAD_115–127 in the context of autologous EBV-transformed B cells. (e) A CD4⁺ T cell clone sorted from the islets from donor T1D.7 recognized IA-2_545–562 in the context of autologous EBV-transformed B cells. Priess B cells were transduced with lentiviral vectors containing open-reading frames (ORFs) of autoantigens were used as antigen-presenting cells (Supplementary Fig. 4 and Online Methods). The autoantigens represented by the ORFs were myelin oligodendrocyte glycoprotein (MOG), GAD65, pre-proinsulin, zinc transporter 8 (ZNT8), myelin basic protein (MBP) and chromogranin A (ChgA). (f) A CD4⁺ T cell line grown from an islet from donor nPOD6323 reacted with a Priess B cell transduced with the ChgA ORF. (g,h) Two CD4⁺ T cell lines grown from separate islets from donor nPOD69 reacted with a Priess B cell transduced with a pre-proinsulin ORF. (i) A CD4⁺ T cell line grown from an islet from donor nPOD69 reacted with a Priess B cell transduced with the ChgA ORF. Three CD8β⁺ T cell lines were grown from handpicked islets on a gel matrix from donor nPOD6268. (j–l) After two rounds of polyclonal stimulation and expansion with cytokines, lines were stained with pools of HLA-A2 pentamers loaded with peptides insulin B_10–18, IA-2_797–805 and IGRP_265–273 (j) or GAD_114–123, PPI_15–24 and IAPP_5–13 (k) and line 6268.6 (l) stained with an HLA-A2 pentamer loaded with a malaria circumsporozoite protein_319–327 as a negative control. One of three similar experiments is shown with data presented as the mean ± s.d. (SD) of triplicates of cell culture wells. P values as determined by two-tailed paired Student’s t-tests and 95% confidence intervals are shown in the figures.

Figure 3

Detection of autoreactivity of CD4⁺ T cell lines and clones sorted or directly grown from islets from donors with T1D with modified peptides. CD4⁺ T cell lines and clones were tested for autoreactivity with panels of peptides synthesized with substitutions corresponding to post-translational modifications with co-cultures, as described in Figure 2. (a) A CD4⁺ T cell line grown from an islet from donor nPOD6323 responded to an HLA-matched, EBV-transformed B cell (DR3⁺, DR4⁺, DQ2⁺, DQ8⁺) pulsed with GRP78_292–305 with an arginine (Arg)-to-citrulline (Cit) modification at amino acid position 297. (b) A CD4⁺ T cell clone sorted directly from islets from donor T1D.7 secreted IFN-γ in response to an autologous EBV-transformed B cell line pulsed with IAPP_65–84 with two Arg-to-Cit modifications at amino acid positions 73 and 81. (c) A CD4⁺ T cell line grown directly from an islet from donor nPOD6367 responded to HLA-matched, EBV-transformed B cells pulsed with a hybrid insulin peptide (insulin C peptide and insulin A chain, hEGGG:A chain—GQVELGGG:GIVEQCC). (d) A CD4⁺ T cell line grown directly from an islet from donor nPOD6323 secreted IFN-γ in response to HLA-matched, EBV-transformed B cells pulsed with hybrid insulin peptide (insulin C peptide and IAPP, hEGGG:IAPP1 (GQVELGGG: TPIESHQ). (e) A CD4⁺ T cell line grown directly from an islet from donor nPOD6323 secreted IFN-γ in response to HLA-matched, EBV-transformed B cells pulsed with hybrid insulin peptide (insulin C peptide and IAPP, hEGGG:IAPP2 (GQVELGGG:NAVEVLK). One of three similar experiments is shown, with data presented as the mean ± s.d. of triplicates of cell culture wells. P values shown are determined by two-tailed paired Student’s t-tests and 95% confidence intervals.