Fig. 2: Crinophagy inhibition diminishes in vivo insulin presentation and reduces the epitope repertoire in pancreatic islets.
a Presentation of G20 by dispersed islet cells (islet resident macrophages) from 6-week-old male NOD mice given PBS or chloroquine without antigen pulse or during pulse with the B:9–23 peptide (10 µM). Data (mean ± s.e.m) summarize results from three independent experiments; each symbol represents one biological replicate, with 2–5 mice pooled together (for the no antigen pulse group, n = 30; for the peptide pulse group, n = 8). ns, not significant; **P = 0.0022; Mann–Whitney test, two-tailed. A schematic figure is created in BioRender. Wan, X. (2024) BioRender.com/s20b261. b Spontaneous presentation of G20 by blood leukocytes isolated from 6-week-old male NOD mice given PBS or chloroquine without antigen pulse or during pulse with the B:9–23 peptide (10 µM). The specificity of MHC-II presentation was tested by adding the anti-I-Ag7 blocking antibody to the culture. Data (mean ± s.e.m) summarize results from four independent experiments; each symbol represents results from an individual mouse (n = 8). ns not significant; ***P = 0.0002; Mann–Whitney test, two-tailed. A schematic figure is created in BioRender. Wan, X. (2024) BioRender.com/s20b261. c Schematic for assessing the crinosome peptidome, created in BioRender. Wan, X. (2024) BioRender.com/s20b261. Each condition (PBS or chloroquine) included 1.2 × 106 live beta cells from ~1500 islets purified from 10 mice. d The number of individual peptide sequences at indicated lengths identified in the peptidome of the crinosomes subcellular fraction from islets of NOD mice given PBS or chloroquine. ****P < 0.0001; two-way ANOVA analysis. e Relative abundance of individual peptides (each symbol) belonging to indicated β-cell secretory proteins in the crinosome peptidomes (mean ± s.e.m). The comparisons were made between PBS and Chloroquine groups within each individual protein. **P = 0.0014; ****P < 0.0001; Wilcoxon test, two-tailed. The numbers of peptides from each protein are Insulin B-chain: 24; C-peptide: 261; ChgA: 133; IAPP: 79; SCG1: 73; SCG2: 131; SCG3: 60. f Relative abundance of individual HIPs (each symbol) in the crinosome peptidomes. ***P = 0.0010; Wilcoxon test, two-tailed. g PTM analysis in the crinosome peptidomes. Denoted are several PTMs with a higher presence in the crinosome peptidome from PBS-treated mice versus those given the chloroquine treatment. h Relative abundance of individual deamidated C-peptides (each symbol) in the crinosome peptidomes (mean ± s.e.m). **P = 0.0018; Wilcoxon test, two-tailed. Number of peptides detected: 11.
