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I-Ag7 β56/57 polymorphisms regulate non-cognate negative selection to CD4+ T cell orchestrators of type 1 diabetes

Nature Immunology volume 24, pages 652–663 (2023)Cite this article

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Abstract

Genetic susceptibility to type 1 diabetes is associated with homozygous expression of major histocompatibility complex class II alleles that carry specific beta chain polymorphisms. Why heterozygous expression of these major histocompatibility complex class II alleles does not confer a similar predisposition is unresolved. Using a nonobese diabetic mouse model, here we show that heterozygous expression of the type 1 diabetes-protective allele I-Ag7 β56P/57D induces negative selection to the I-Ag7-restricted T cell repertoire, including beta-islet-specific CD4+ T cells. Surprisingly, negative selection occurs despite I-Ag7 β56P/57D having a reduced ability to present beta-islet antigens to CD4+ T cells. Peripheral manifestations of non-cognate negative selection include a near complete loss of beta-islet-specific CXCR6+ CD4+ T cells, an inability to cross-prime islet-specific glucose-6-phosphatase catalytic subunit-related protein and insulin-specific CD8+ T cells and disease arrest at the insulitis stage. These data reveal that negative selection on non-cognate self-antigens in the thymus can promote T cell tolerance and protection from autoimmunity.

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Fig. 1: Pancreas targeting by islet-specific T cells and T1D requires exclusive I-Ag7 expression.
Fig. 2: I-Ag7 β56/57 polymorphisms regulate thymocyte negative selection at the double-positive stage.
Fig. 3: I-Ag7-PD manifests T cell tolerance on the I-Ag7-restricted T cell repertoire.
Fig. 4: I-Ag7-PD induces non-cognate negative selection of tetramerhi islet-specific CD4+ T cells.
Fig. 5: Non-cognate negative selection limits islet-specific CXCR6+ CD4+ T cell development.
Fig. 6: Loss of insulin and IGRP CD8+ T cell cross-priming in I-Ag7-PD-expressing mice.
Fig. 7: CXCR6+ CD4+ T cells orchestrate islet-specific CD8+ T cell cross-priming and T1D.

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Data availability

TCR sequencing data have been deposited in the NIH Sequence Read Archive under the accession codes: SRR16314909, SRR16314908,

SRR16314907, SRR16314905, SRR16314906, SRR16314942, SRR16314941, SRR16314940,

SRR16314939, SRR16314938, SRR16314937, SRR16314936, SRR16314935, SRR16314934,

SRR16314933, SRR16314932, SRR16314931, SRR16314926, SRR16314925, SRR16314924,

SRR16314930, SRR16314929, SRR16314928, SRR16314927, SRR16314923, SRR16314946,

SRR16314945, SRR16314944, SRR16314943, SRR16314921, SRR16314920, SRR16314919,

SRR16314918, SRR16314917, SRR16314916, SRR16314915, SRR16314922, SRR16314914,

SRR16314913, SRR16314912, SRR16314911, SRR16314910, SRR16313009, SRR16313011, SRR16313010, SRR16313008, SRR16313013, SRR16313012, SRR16313057, SRR16313056,SRR16313062, SRR16313061, SRR16313060, SRR16313059, SRR16313058, SRR3723003, SRR3723002, SRR3722997, SRR3722996, SRR3722995, SRR3722994, SRR3722993 and SRR3722992.

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Acknowledgements

Supported by the US National Institutes of Health (NIH; AI143976 and AR071269) to E.S.H. S.B.C. was supported by an NIH training grant (5T32A1007349-31).

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Authors and Affiliations

  1. Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA

    Brian D. Stadinski, Sarah B. Cleveland, Priya G. Huseby & Eric S. Huseby

  2. Department of Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA

    Michael A. Brehm & Dale L. Greiner

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  1. Brian D. Stadinski
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Contributions

B.D.S. and E.S.H. conceived and designed the project and interpreted experiments; B.D.S. performed TCR cloning and sequencing, flow cytometry, adoptive T cell transfer experiments, monitored disease and statistical analyses; S.B.C. performed histological analyses and T cell activation studies; M.A.B. and D.L.G. provided peripheral blood mononuclear cells from HLA-typed individuals with and without T1D; P.G.H. generated specific mouse lines and monitored disease; B.D.S. and E.S.H. wrote the manuscript.

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Correspondence to Eric S. Huseby.

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Nature Immunology thanks Ludger Klein, Maki Nakayama and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characterization of NOD mice carrying different combinations of I-Ag7 b56/57 polymorphisms.

(a) Genomic DNA sequence surrounding I-Ag7 b56/57 of NOD mice carrying the wild type (HS), PD or frame shift (KO) mutation. (b) MHC-II expression on B cells from I-Ag7-WT/WT (n = 11), I-Ag7-KO/WT (n = 9) and I-Ag7-KO/KO (n = 8) mice. (c) T1D incidence in I-Ag7-WT/WT mice derived from intercrossing I-Ag7-PD/WT or I-Ag7KO/WT mice. ns P > 0.05, logrank Mantel-Cox test. (d) T1D incidence in I-Ag7-KO/KO and I-Ag7-WT/WT mice derived from intercrossing I-Ag7-KO/WT mice. (e–h) Cell surface expression of MHC molecules on APC subsets. (e,f) I-Ag7 and (g,h) H2-Kd expression level and average mean fluorescence intensity (MFI) of CD19+ B cells, CD11c+ XCR1neg cDC2 and CD11c+ XCR1pos cDC1 from I-Ag7-WT/WT (n = 5) and I-Ag7-PD/PD (n = 5) mice. (i,j) Representative hematoxylin and eosin staining of a grade 3 islet and grade 1 islet from the pancreas from I-Ag7 WT/WT and I-Ag7 PD/WT bearing mice, respectively. (k) Average frequency of islet infiltration severity at 12 weeks of age (n = 8) using a four scale grade: 0 - insulitis free, 1- peri-insulitis, 2 – moderate < 50% infiltrated, 3 – severe > 50% infiltration. (l) Quantification of Foxp3+ regulatory T cell subsets within the pancreatic infiltrate of I-Ag7 WT/WT (n = 24), I-Ag7 KO/WT (n = 10), I-Ag7 PD/WT (n = 25) or I-Ag7 PD/PD (n = 10) NOD mice. n = biologically independent animals. ns P > 0.05; * P < 0.05; ** P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test).

Extended Data Fig. 2 Characterization of thymic T cell selection in 6 weeks old I-Ag7-WT/WT, I-Ag7-KO/WT, I-Ag7-PD/WT and I-Ag7-PD/PD mice.

(a) Total number of CD4−CD8− and (b) semimature (SM), mature 1 (M1) and mature 2 (M2) CD4 SP thymocyte generated in each strain. (c) Percent of CD4SP and total number of Foxp3 + CD4SP thymocytes generated in each strain. (d–e) Frequencies of TCR Vβ+ T cells among (d) CD4+ and (e) CD8+ subsets in I-Ag7-WT/WT, I-Ag7-PD/WT and I-Ag7-PD/PD mice. (f) Representative dot plots of CD69 and Nur77-GFP expression on preselection TCRβ+ DP thymocytes derived from 4−6 weeks old Nur77-gfp+ H2-Ab1−/− B2m−/− (MHCdeficient) mice following co-culture with BM-DCs generated from I-Ag7-WT/WT, I-Ag7-PD/PD or C57BL/6 (H-2b) mice. (g) Quantification of the frequency and (h) percent of H-2g7 reactivity at which DP thymocytes from 6 mice express CD69 and Nur77-GFP following culture with BM-DC from 2 independent mice. Data are from 2 independent experiments. (i) Representative dot plots of CD69 and Nur77-GFP expression on pre-selection TCRβ+ DP thymocytes (n = 6) derived from 4–6 weeks old Nur77-gfp+ H2-Ab1−/− B2m−/− (MHC-deficient) mice following co-culture with BMDCs generated from 3 b2M−/− I-Ag7-WT/WT, b2M−/− I-Ag7-PD/PD or b2M−/− I-Ag7-KO/KO mice. (j) Quantification of the frequency and (k) percent of I-Ag7 reactivity at which DP thymocytes express CD69 and Nur77-GFP following culture with BM-DC. Data are from two independent experiments. P values are from a one-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 3 HLA analyses and representative flow cytometry sorting of donors with T1D and healthy controls.

(a) HLA status of donors, including positivity or negativity for HLA-A2, HLA-DR3, HLA-DR4, and HLA-DQ usage. (b) Example of flow cytometry sorting of human samples for isolation of naïve CD4 and naïve CD8 T cells. All TCR+ lymphocytes were sorted based on being a single cell that expresses CD3 and CD4 or CD8. CD4 Tconv cells were further sorted for the expression of CD25 and CD127, and CD27, CD45RO, CCR7 and CD95. CD8 Tconv were further sorted for the expression of CD27, CD45RO, CCR7 and CD95.

Extended Data Fig. 4 Expression of I-Ag7-PD induces negative selection of tetramerbright IAg7-ChgAHIP reactive thymocytes at the DP to SP transition.

(a) Flow cytometric analysis and quantification of IAg7-ChgAHIP tetramer staining of CD4SP thymocytes following tetramer-based enrichment, isolated from I-Ag7-WT/WT (n = 6), I-Ag7-PD/WT (n = 6) mice or BDC2.5 TCR Tg on an IAg7-WT/WT genetic background (n = 7), or polyclonal CD4SP thymocyte without enrichment. (b) Mean fluorescence intensity (MFI) of tetramerbright and tetramerdim populations of CD4SP thymocytes for each mouse strain. (c–h) BDC2.5 TCR Tg thymocytes undergo negative selection at the DP to CD4SP transition. (c–h) Flow cytometric analysis of CD4 and CD8−expressing thymocyte subsets from 6 weeks old BDC2.5 TCR Tg mice on a I-Ag7-WT/WT (n = 7), I-Ag7-PD/WT (n = 7) or I-Ag7-PD/PD (n = 7) genetic backgrounds, and (d) quantification of CD4SP thymocyte frequency. (e) Representative examples and (f) quantification of CD4+CD8+ thymocytes expressing CD69 and high levels of TCRβ. (g) Representative examples and (h) quantification of frequency at which BDC2.5 TCR Tg mice matured thymocytes into T cells, based on expression of MHCI and high levels of TCRb. P values are from a one-way ANOVA with Tukey’s multiple comparisons test. n = biologically independent animals.

Extended Data Fig. 5 Exclusive expression of I-Ag7-WT in bone marrow derived cells allows the development of tetramerbright IAg7-ChgAHIP reactive T cells in I-Ag7-WT/WT and I-Ag7-PD/WT hosts.

(a) Representative staining from concatenated data (n = 2 mice) of CD4SP thymocytes from radiation chimeras of I-Ag7-WT/WT bone marrow (BM) into I-Ag7-WT/WT, I-Ag7-PD/WT, I-Ag7-PD/PD or H2b expressing mice. (b) Overlay of I-Ag7-ChgAHIP tetramer+ CD4SP thymoctes from I-Ag7-WT/WT and IAg7-PD/WT chimeras with I-Ag7-WT/WT BM. (c,d) Quantification of the (c) number and (d) percentage of I-Ag7-ChgAHIP tetramerbright and tetramerdim cells among individual I-Ag7-WT/WT BM chimeric mice. (e) Overlay and (f,g) MFI of I-Ag7 staining on cortical and medullarly thymic epithelial cells of 6 weeks old I-Ag7-WT/WT (n = 5), I-Ag7-PD/WT (n = 4), I-Ag7-PD/PD (n = 5) mice. (h–j) Example and quantification of IL-2 release from BDC2.5 T cells hybridomas with co-cultured with ChgAHIP peptide and sorted (i) cTECs and (j) mTECs populations from I-Ag7-WT/WT, I-Ag7-PD/WT, I-Ag7-PD/PD mice. Example (k) contour plot and (l) plotted average % CTVdim BDC2.5 CD4 T cells proliferating in response to BM-DCs derived from I-Ag7-WT/WT and I-Ag7-PD/PD presenting β-islet cell preparations based on from 3 replicate wells per experiment using separate islet preparation per experiment. (m,n) Quantification of IL-2 release by BDC2.5 and BDC6.9 T cell hybridomas in response to BM-DCs derived from I-Ag7-WT/WT and I-Ag7-PD/PD presenting β-islet cell preparations or soluble peptide. Bars are mean values from the 6 wells. P values are from a one-way ANOVA with Dunnett’s multiple comparisons test.

Extended Data Fig. 6 Expression of I-Ag7-PD does not result in the differentiation of islet-specific CD4 T cells into Foxp3+ Tregs.

(a,b) Flow cytometric analysis of CD44 and VLA4 on (a) I-Ag7-ChgAHIP- and (b) I-Ag7-IAPPHIPspecific CD4 T cells. (c,d) Flow cytometric analysis of Foxp3-gfp and CD25 on (c) I-Ag7-ChgAHIP- and (d) I-Ag7-IAPPHIP-specific CD4 T cells. Quantification of (e,h) CD44 and VLA4 expression, (f,i) FR4 expression and (g, j) Foxp3-gfp expression on (e–g) I-Ag7-ChgA and (h–j) I-Ag7-IAPP specific CD4 T cells isolated from I-Ag7-WT/WT (n = 13, 6), I-Ag7-KO/WT (n = 6, 6), I-Ag7-PD/WT (n = 15, 7) and I-Ag7-PD/PD (n = 9, 6) mice, respectively. ns P > 0.05; * P < 0.05; ** P < 0.01; **** P < 0.0001 (oneway ANOVA with Tukey’s multiple comparisons test). (k,l) Activation and quantification of EC50 values of 8 ChgAHIP-reactive T cell hybridomas in response to titrating amounts of ChgAHIP peptide co-cultured with I-Ag7-WT/WT, I-Ag7-PD/WT, I-Ag7-PD/PD splenocytes. (m,o) I-Ag7-ChgAHIP tetramer binding of (m) T cell hybridomas 80.B2 (red), 80.C1 (orange) and 82.B1 (blue), (n) T cell transfectomas 80.B2 (red), 80.C1 (orange) and 82.B1 (blue). T cell hybridomas, and T cell transfectomas expressed similar levels of TCR. (o) TCR Vα and Vβ sequences from I-Ag7-ChgAHIPreactive hybridomas isolated from I-Ag7-WT/WT, I-Ag7-PD/WT mice. (p) Influence of TCR:pMHC affinity and antigen concentration on CD4 T cell effector functions. B3K506 TCR T cells were activated with titrating concentrations of strong (3K; KD = 7 mM), medium (P-1A; KD = 26 mM), weak (P8A; KD = 92 mM) and very weak (P2A; KD = 278 mM) affinity ligands and expression of CD40L, CXCR6, VLA4 and FR4 was evaluated, as well as cellular proliferation.

Extended Data Fig. 7 I-Ag7-PD expressing mice can expand high frequencies of Kd-IGRP206–214 and Kd-insulin15–23 specific T CD8 cells following viral activation.

(a,b) Flow cytometric analysis and quantification of Kd-IGRP206–214 and (c,d) Kd-insulin15–23 specific CD8 T cells following tetramer-based enrichment, isolated from I-Ag7-WT/WT (n = 9, 9), IAg7-PD/WT (n = 6, 9) mice infected with VSV-IGRP206–214 or VSV-Insulin15–23, respectively. (e) Flow cytometric analysis and (f) quantification of CD40L expression on CXCR6+ and FR4+ CD4 T cells from BDC2.5 TCR Tg mice (n = 4) following 4hrs in vitro stimulation with anti-CD3/CD28. ns P > 0.05; **** P < 0.0001 (unpaired two-tailed t-test). (g) Purity of flow cytometry sorting of mouse CXCR6+ and FR4+ T cells for adoptive transfer. n = biologically independent animals.

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Table of mouse and human antibodies used.

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Stadinski, B.D., Cleveland, S.B., Brehm, M.A. et al. I-Ag7 β56/57 polymorphisms regulate non-cognate negative selection to CD4+ T cell orchestrators of type 1 diabetes. Nat Immunol 24, 652–663 (2023). https://doi.org/10.1038/s41590-023-01441-0

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