Fig. 6: CD4⁺ T cells reactive to unconventional epitopes derived from crinosomes exhibit a nontolerant phenotype associated with pathogenicity.

a Flow cytometry analysis of peripheral tetramer-binding CD4⁺ T cells for expression of FOXP3 and CD44. The quantification (right) summarizes the percentage of the FOXP3⁺ Treg population in tetramer-positive epitope-specific and tetramer-negative (Tet-) polyclonal CD4⁺ T cells. Data (mean ± s.e.m) summarize results from 4 to 6 independent experiments; each symbol represents results from an individual mouse (n = 8: CP2, n = 9: E21, n = 4: DBP, n = 8: ZnT8, n = 5: CP1d, n = 9: G20, n = 6: 2.5HIP, n = 7: 6.9HIP, n = 37: polyclonal T cells). ****P < 0.0001; Two-way ANOVA analysis. b Flow cytometry analysis of FOXP3⁺ Tregs in E21:I-A^g7 and G20:I-A^g7 tetramer-binding CD4⁺ single-positive thymocytes. The bar graph quantifies the percentage of FOXP3⁺ cells among the tetramer-binding CD4⁺ T cells. Data (mean ± s.e.m) summarize results from three independent experiments; each symbol represents results from an individual mouse (n = 7). ***P = 0.0006; Mann–Whitney test, two-tailed. c ELISPOT assay for assessing T cell responses upon recall with Syn-E21 (TEGEALYLVCGEGGS) or Syn-G20 (TEGVEALYLVCGGGS) in NOD treated with different blocking antibodies and immunized with the B:9–23 peptide (n = 10 for untreated group, n = 10 for anti-TGFβ treated group, n = 4 for anti-CTLA4 treated group, n = 6 for anti-CD25 treated group). Data (mean ± s.e.m) summarize results from three independent experiments; each symbol represents results from an individual mouse. **P = 0.0028; ***P = 0.0004; ****P < 0.0001; Mann–Whitney test, two-tailed. The schematic figure is created in BioRender. Wan, X. (2024) BioRender.com/y60m085. d ELISPOT assay for assessing T cell responses upon recall with each individual epitope in the thymic or islet epitope set after a two-cycle in vitro stimulation of islets and pLN cells with the same peptides. Data (mean ± s.e.m) summarize results from 2 to 5 independent experiments; each symbol represents a biological replicate including 4–6 mice pooled together. (n = 20 for CP1d and G20 in 5 independent experiments, n = 8 for 2.5HIP and 6.9 HIP in 2 independent experiments, n = 18 for CP2 and E21 in 3 independent experiments, n = 20 for DBP and ZnT8 in 4 independent experiments) ****P < 0.0001; Two-way ANOVA analysis. e ELISPOT assay for assessing T cell responses upon recall with each individual thymic epitope after a two-cycle in vitro stimulation of islets and pLN cells pooled from NOD mice treated with the control or the anti-CD25 antibody. Data (mean ± s.e.m) summarize results from multiple independent experiments; each symbol represents a biological replicate including 4–6 mice pooled together. (n = 20 for CP2, E21, and ZnT8 in 4 independent experiments; n = 16 for DBP in 3 independent experiments). *P = 0.0351; **P = 0.001; ***P = 0.0002; Repeated measures two-way ANOVA with Bonferroni’s multiple comparisons test. The schematic figure is created in BioRender. Wan, X. (2024) BioRender.com/s30d279.