Abstract
Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12–20 segment of the insulin B-chain (B:12–20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13–214. CD4 T cells that recognize B:12–20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13–21 have only a minor role in autoimmunity3,4,5. Although presentation of B:12–20 is evident in the islets3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in β-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.
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Acknowledgements
We thank J. Carrero, A. Shaw and F. Urano for critical support and review of the work; and B. Saunders, W. Beatty, H. Hu and K. Fredericks for technical assistance. This work was supported by National Institutes of Health grants DK058177 and AI14551. The laboratory also received generous support from the Kilo Diabetes & Vascular Research Foundation.
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Nature thanks S. Barg, P. Santamaria and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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X.W. and E.R.U. conceived the project, designed the experiments and analysed the results. B.H.Z. and X.W. performed two-photon microscopy and analysed the data. A.N.V. and X.W. prepared samples for mass spectrometry. C.F.L. performed the nLC–MS/MS studies and analysed the data. A.N.V. generated monoclonal antibodies and isolated β-cell granules. X.W. and A.N.V. performed cell biology experiments. P.N.Z. and X.W. analysed the RNA-seq data. M.S.A., R.T. and L.S. provided key reagents and interpreted the results. X.W., C.F.L. and E.R.U. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Probing peripheral antigen presentation by two-photon imaging; the motility assay.
a, Representative 3D reconstructions of two-photon z-stacks visualizing CFSE-labelled anti-HEL 10E11 TCR transgenic and CMTMR-labelled wild-type CD4 T cells in an iLN explant on day 3 post transfer. Individual T cells were tracked in the area bound by the dashed line. Right, magnified views of this region, showing movement of T cells over a 7.5-min time interval. Quantification was performed over a 5-min interval. Cyan and purple tracks represent 10E11 and wild-type T cells, respectively. Mice were injected with 10 µg HEL. b, NOD mice (CD45.1) were injected intraperitoneally with indicated amounts of HEL. Six hours after injection, naive CFSE-labelled 10E11 (CD45.2) T cells were transferred. On day 3, CFSE dilution of the transferred T cells (CD45.2+CD45.1–CD4+Vβ8.1/8.2+) in the iLNs was measured by flow cytometry. Data are representative of two independent experiments. c, Mean track velocities of 8F10 and wild-type CD4 T cells in iLNs from NOD recipients on day 1 or day 5 post transfer. d, CFSE(8F10) plus CMTMR(WT) or CMTMR(8F10) plus CFSE(WT) T cells were separately transferred into two cohorts of NOD recipients, and their mean track velocities in iLNs on day 3 were compared by paired two-photon imaging analysis. e, Mean track velocities of 8F10 and wild-type CD4 T cells in NOD.µMT or NOD.Batf3–/– recipients on day 3 post transfer. f, Mean track velocities of 8F10 and 10E11 T cells in NOD.H2b recipients 24 h post transfer. g, h, Mean track velocities of 4F7 and wild-type CD4 (g) or 8.3 and wild-type CD8 (h) T cells in NOD recipients on day 3 post transfer. i, Response (mean ± s.e.m.) of the B:13–21-specific IIT-3 T cells to ConA-activated peritoneal macrophages treated with or without S961 before insulin pulse. j, Blood glucose levels (mean ± s.e.m.) of 3-week old NOD mice infused with S961 or PBS via osmotic pumps. k, The scheme of the experiments in Fig. 1h, i. l, Mean track velocities of 8F10 and wild-type CD4 T cells in iLNs of Aire–/– recipients. Data summarize two (c, d, f, l) or three (e, g, h) independent experiments. Each dot represents individual T cell tracks, and the bar denotes the mean. ns, not significant; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test (c, d, g, h) or two-tailed unpaired Student’s t-test (e, f, l).
Extended Data Fig. 2 Analysis of insulin peptide-specific monoclonal antibodies and presentation of the intact B-chain.
a–c, Competitive ELISA responses showing the binding of: anti-insulin monoclonal antibody (E11D7) to plate-bound insulin (a) anti-B:9–23 monoclonal antibody (AIP) to plate-bound B:9–23 (b), and anti-B:1–30 monoclonal antibody (6F3.B8) to plate-bound B:1–30 (c) in the presence of serial dilutions of the indicated soluble antigens as a competitive inhibitor. Inhibition by a specific soluble antigen indicates the specificity of the monoclonal antibody to this antigen. d, Competitive ELISA responses showing the binding of 6F3.B8 to plate-bound B:1–30 in the presence of soluble unmodified B:1–30 or B:1–30 in which the two cysteines were changed to serines (B:1–30 C to S). The results indicate the intrachain link formed by the cysteines does not influence the specificity of the 6F3.B8 monoclonal antibody. Data are means representing two independent experiments. e, Responses of the B:13–21-specific IIT-3 (left) or the B:12–20-specific 9B9 (right) T cell hybridoma to C3g7 APCs treated with or without 100 µM chloroquine for 2 h and pulsed with indicated antigens after extensive washes. C3g7 cells are a B cell lymphoma line expressing I-Ag7, and are used as APCs. The results of the effects of chloroquine indicate that reactivity to insulin, but not to B:9–23 or B:1–30 require internal processing. Data are mean ± s.e.m., representative of two independent experiments.
Extended Data Fig. 3 nLC–MS/MS analysis of mouse β-cell granules.
a, Mass spectra of mouse insulin-1 B:1–30 with intramolecular disulphide bonds (left) and mouse insulin-2 B:1–30 with oxidized methionine in position 29 (right). b, Mass spectra of mouse insulin B:9–23 (left) and B:11–23 (right), which were exclusively identified in the 5k granules of B6g7, B6 and NOD mice. c, Mass spectra of two hybrid peptides identified in the 5k granules. The sequence (EVEDTPVRSGSNPQM, left) represents a C-peptide (underlined)–islet amyloid polypeptide (IAPP) fusion, and the sequence (EVEDPQVAEVARQ, right) represents a fusion of the N terminus of insulin-2 C-peptide (underlined) with the C terminus of insulin-1 C-peptide.
Extended Data Fig. 4 nLC–MS/MS analysis of human β-cell granules.
a, Peptide coverage of insulin B chain identified in human 25k (red) and 5k (blue) β-cell granules using nLC–MS/MS analysis. Shown is the alignment of individual peptides (each line) with the human insulin B:1–30 segment. Data summarizes results from four independent runs using human islets from three individual donors. b, A mass spectrum showing a sequence representing human insulin B:11–30 that was identified in the 5k granules. The cysteinylation in position 19 is indicated.
Extended Data Fig. 5 Analysis of insulin peptides secreted from islets upon glucose challenge.
a, Insulin secretion assay was performed as described in Fig. 3a–c, except that protease inhibitors were added during the 25-mM glucose challenge. The supernatants were then collected for the competitive ELISA assay. Data are mean ± s.e.m. from two independent experiments. b, Mass spectra of four secreted peptides that contain the B:12–20 and/or B:13–21 epitopes as listed in Fig. 3e. Secreted B:1–30 sequences are identical to those in Extended Data Fig. 3a, and B:9–23 and B:11–23 share identical sequences with those in Extended Data Fig. 3b. c, A mass spectrum of the secreted insulin B:15–23 MHC-I (Kd)-binding peptide. d, A mass spectrum of the secreted insulin A:14–20 MHC-I (Db)-binding peptide. e, A mass spectrum showing a representative B–C-spanning peptide (B25–C23).
Extended Data Fig. 6 T cell responses to B:9–23-associated peptides.
Responses of three insulin-reactive T cell hybridomas to insulin peptides associated with the 9–23 region of the B chain as identified in Fig. 3e. The C3g7 cells were used as APCs. Data are mean ± s.e.m.
Extended Data Fig. 7 Characterization of circulating B:9–23 and its localization into lymphoid organs.
a, Unmodified synthetic B:9–23 (3 pmol) was spiked into 1 ml PBS, purified using C18 tips, lyophilized, and analysed by nLC–MS/MS. The data show the appearance of unmodified B:9–23 (left) together with oxidation of Cys19 to cysteic acid (right). b, c, Alexa Fluor 488-conjugated B:9–23 peptide (100 µg) was injected intravenously into 4-week old B6, B6g7 and NOD mice. An hour later, spleens and thymi were harvested, digested with liberase and DNase, and binding to splenic and thymic APCs was measured by flow cytometry. b, Representative FACS plots showing the binding of B:9–23 to splenic XCR1+ and Sirpα+ dendritic cell (DC) subsets and B cells (top). The bar graph summarizes cumulative results from individual mice (each point), pooled from three independent experiments. ns, not significant; **P < 0.05; ***P < 0.01; ****P < 0.005, two-tailed unpaired Student’s t-test. c, Representative FACS plots showing the binding of B:9–23 to thymic XCR1+ and Sirpα+ DC subsets and to CD45– cells expressing MHCII. Data are mean ± S.D from five individual mice per strain from two independent experiments.
Extended Data Fig. 8 RNA-seq analysis of 8F10 T cells developed in NOD or B16A hosts.
a, Representative FACS plots (top) showing the sorting strategy and recovery of 8F10 T cells from iLNs of NOD or B16A-recipient mice six weeks after adoptive transfer of bone marrow. The scatter plot (bottom) shows the percentage of recovered 8F10 T cells among total CD4 T cells from four independent experiments. ns, not significant; two-tailed paired Student’s t-test. b, Biological pathways that are significantly enriched in the 8F10-NOD versus 8F10-B16A samples using GSEA and Hallmark database. c, Heat maps of all enriched genes in individual metabolic pathways depicted in Fig. 4c.
Extended Data Fig. 9 8F10 T cells exhibit an effector phenotype, but no anergy or exhaustion phenotype, at the transcription level during peripheral antigen recognition.
a, Heat maps showing all the enriched genes of the three immunological pathways illustrated in Fig. 4d. b, GSEA enrichment plots performed on differentially expressed genes in 8F10 T cells from the NOD-iLN versus B16A-iLN condition using datasets characterizing CD4 T cell anergy and CD8 T cell tolerance.
Extended Data Fig. 10 Functional analysis of 8F10 T cells developed in NOD or B16A hosts.
a–f, The bone marrow chimaera was constructed as in Fig. 4a, and T cells were examined after 6 (a–c) or 9 (d–f) weeks. a, b, d, e, Bulk CD4+ T cells were purified from iLNs of individual NOD or B16A mice (three per group) by two rounds of MACS negative selection. To examine cytokine repertoire (a, d), half of the individual T cell samples were combined. The remainder were kept as individual samples, labelled with CFSE (1.5 µM), and used to measure cell proliferation (b, e). In either case, T cells were mixed with NOD.Rag1–/– splenocytes (1:2 ratio) and stimulated with B:9–23 for 16 (a, d) or 72 (b, e) hours. a, Representative FACS plots showing intracellular cytokine staining of the 8F10 T cells from NOD-iLN or B16A-iLN, after stimulation with B:9–23 for 16 h (brefeldin A was added for the last 4 h). Production of IL-4, IL-17A, IL-5 and IL-10 was not detected. Data are representative of two independent experiments with 3 mice combined per experiment. b, Representative FACS plots (top) showing CFSE dilution of the 8F10 T cells stimulated by B:9–23 or the control HEL11–25 peptide for 72 h. The results of 6 individual mice from two independent experiments are summarized in the box plots (bottom). Box plots show the median, box edges represent the first and third quartiles, and the whiskers extend from the minimum to the maximum. **P < 0.01, two-tailed unpaired Student’s t-test. c, Representative FACS plots showing ex vivo surface staining of FR4 and CD73 as well as CD39 and TIGIT on endogenous CD4+ or 8F10 T cells in the iLNs of NOD or B16A mice. Data are representative of three mice analysed in two independent experiments. d–f, Experiments were performed in week 9 following the procedures described in a–c. The data in d–f are from a single experiment.
Supplementary information
Supplementary Table 1
Insulin peptides identified in mouse and human granules.
Supplementary Table 2
Islet secretion insulin peptide list.
Video 1: Motility of anti-HEL 10E11 T cells in mice given HEL
The anti-HEL 10E11 (green) and WT polyclonal (red) CD4 T cells were adoptively transferred into NOD mice given indicated amounts of HEL protein. On day 3 post transfer, the inguinal lymph nodes were harvested and imaged by two-photon microscopy. The videos show a progressive reduction of the 10E11 T cell velocities as the antigen amount increases.
Video 2: Motility of anti-insulin peptide 8F10 T cells in lymph nodes
The insulin peptide-reactive 8F10 (green) and WT (red) CD4 T cells were adoptively transferred into NOD mice. On day 3 post transfer, the pancreatic (pLN), inguinal (iLN), and axillary (aLN) lymph nodes were imaged. The videos depict a reduction of the 8F10 T cell motility relative to the WT controls in all the lymph nodes.
Video 3: Motility of anti-insulin peptide 8F10 T cells in the absence of the antigen
The insulin peptide-reactive 8F10 (green) and WT (red) CD4 T cells were adoptively transferred into B16A mice that lack immunogenic insulin peptides. On day 3 post transfer, the pancreatic (pLN) and inguinal (iLN) lymph nodes were imaged. The videos depict a comparable level of velocities between the 8F10 and WT T cells in the B16A hosts.
Video 4: Motility of anti-insulin 4F7 T cells in mice with in vivo blockade of insulin receptor
The insulin-reactive 4F7 (green) and WT (red) CD4 T cells were adoptively transferred into NOD mice that were infused with PBS or the insulin receptor antagonist S961. On day 3 post transfer, the inguinal (iLN) lymph nodes were imaged. The videos depict a partial reduction of the 4F7 T cell motility influenced by S961 treatment relative to the PBS control.
Video 5: Motility of anti-insulin peptide 8F10 T cells in mice with in vivo blockade of insulin receptor
The insulin peptide-reactive 8F10 (green) and WT (red) CD4 T cells were adoptively transferred into NOD mice that were infused with PBS or the insulin receptor antagonist S961. On day 3 post transfer, the inguinal (iLN) lymph nodes were imaged. The videos depict a comparable level of motility arrest in the 8F10 T cells relative to WT T cells between mice infused with PBS and S961.
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Wan, X., Zinselmeyer, B.H., Zakharov, P.N. et al. Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides. Nature 560, 107–111 (2018). https://doi.org/10.1038/s41586-018-0341-6
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DOI: https://doi.org/10.1038/s41586-018-0341-6
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