Fig. 7

Inhibition of USP7 in tolerogenic Stat5b-CA.DCs reduce Tregs and Th2 responses and promote IL-17-producing Th17 cell subset. Purified splenic DCs from NOD and NOD.Stat5b-CA mice were pre-incubated with (+ PD5091) or without (− PD509) USP7 inhibitor (5 µM/mL) for 24 h and then stimulated with LPS (1 µg/mL) for additional 24 h. Cells were then washed, and 6 × 10⁶ cells were i.v injected into 8–10 weeks old NOD mice. After 7 days, spleen cells were harvested and analyzed for Tregs and Th1/Th2/Th17 cell subsets and their cytokine profiles by FACS. A–H Representative FACS profile and percentage of CD4⁺Foxp3⁺ Tregs (A, B), CD4⁺Gata-3⁺ Th2 (C-D), CD4⁺RORγt⁺ Th17 (E, F) and CD4⁺T-bet⁺ Th1 (G, H) cells. I–L Splenic cells of recipient NOD mice were stained with different T cell subsets and cytokine antibodies and analyzed by FACS. Percentages of Foxp3⁺IL-10⁺ (I), CD4⁺IL-4⁺ (J), CD4⁺IL-17⁺ (K), and CD4⁺IFN-γ⁺ (L) T cell subsets. In all experiments, 4 mice per group were used, and the results are expressed as mean ± SEM. The significance was calculated using One-way ANOVA with Tukey’s post-test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001