Fig. 2: Disease-relevant signals drive C19S transformation via oxidative stress in β cell granules.
a, Schematic (left) for generating CD4+ T cell hybridomas after immunizing NOD mice with the InsB9-23(C19S) peptide. The bar graph (right; mean ± s.e.m.) quantifies responses of individual T cell hybridomas (n = 9) to 0.3 µM native InsB9-23 or InsB9-23(C19S) peptide. b, Responses of the S5 (left) and 9B9 (right) CD4+ T cell hybridomas to serially diluted concentrations of the native InsB9-23 or the InsB9-23(C19S) peptide. c, Schematic of the GAP assay. d, Bar graphs (mean ± s.e.m.) showing responses of the 9B9 or the S5 T cell to crinosome or DCG fractions isolated from unmanipulated MIN6 cells or B6 mouse islets (n = 4). e, Schematic (left) and bar graph (right; mean ± s.e.m.) showing in vivo presentation of C19S insulin peptides in pancreatic islets from 5- and 17-week-old female NOD mice (n = 3). f, Schematic (left) and bar graph (mean ± s.e.m.) showing ELISPOT assay for T cell responses (IFNγ production) in islets and pancreatic lymph nodes during antigen recall with the InsB9-23 or InsB9-23(C19S) peptide (n = 4). g, Bar graphs (mean ± s.e.m.) showing responses of the S5 T cell to the crinosome fraction isolated from MIN6 cells and B6 mouse islets following indicated treatments (n = 3). h, Bar graphs (mean ± s.e.m.) showing responses of the 9B9 T cell to the crinosome fraction isolated from MIN6 cells and B6 mouse islets following indicated treatments (n = 3). i, Bar graphs (mean ± s.e.m.) showing responses of the S5 or 9B9 T cell to crinosome (n = 6) and DCG (n = 5) fractions isolated from MIN6 cells stimulated with TNFα, with or without glutathione or TUDCA. In a, n is the number of individual hybridomas; each dot is one hybridoma. In d, g, h and i, n is the number of biological replicates; each dot is an independent experiment. In e and f, n is the number of independent experiments; each dot is one sample pooled from multiple mice. For statistical analysis, a two-tailed paired t-test was performed for a. Repeated measures (RM) one-way ANOVA with Tukey’s multiple comparisons test was performed for f. RM one-way ANOVA with Sidak’s multiple comparisons tests were performed for d, g and h. RM two-way ANOVA with Sidak’s multiple comparisons test was performed for e. RM two-way ANOVA with Dunnett’s multiple comparisons test was performed for i. The data represent three (e, g, h), four (d, f) and five (a, i) independent experiments. Illustrations in a, c and e were partly created with BioRender.com.
