Fig 2: The related mechanism by which miR-146a regulates Tregs.A Expression of the TRAF6, IRAK1, and Foxp3 mRNAs in Tregs isolated from the WT mouse or CKO mouse spleens, n = 3. B Levels of the total TRAF6, IRAK1, and Foxp3 proteins and phosphorylated IκB and p65 proteins in Tregs isolated from the WT mouse or CKO mouse spleens, n = 3. C Levels of the total TRAF6, IRAK1, and Foxp3 proteins and phosphorylated IκB and p65 proteins in Tregs isolated from recipient’s spleens were detected on day 5 in the WT, WT+RAPA, CKO, and CKO+RAPA groups. n = 3. D The levels of the mRNA and E total and phosphorylated STAT1 protein in Tregs isolated from the WT or CKO mouse spleens. n = 3. F–G The total protein level and phosphorylation of STAT1 in Tregs isolated from the recipient’s spleens were analyzed on day 5 in the WT, WT+RAPA, CKO, and CKO+RAPA groups, n = 3. H CD4+ T cells from BALB/c spleens were co-cultured with Tregs isolated from the spleens of WT recipients or CKO recipients and treated with control IgG or αIFN-γ for 72 h, and then a flow cytometry analysis of the proportions of Th1 cells was performed. n = 3. I Kaplan-Meier analysis of the allograft survival time (log-rank test). n = 6. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. CKO indicates miR-146a conditional knockout mice. WT wild type, RAPA rapamycin.

Fig 3: JQ1 reduces tumor-infiltrating Foxp3+ regulatory T cells. (A) Comparison of tumor volumes from C57BL/6 mice. n=5; (B) Flow cytometry gating strategy for analyzing Treg cells; (C) Percentage of Foxp3+ Treg cells among CD4+ T cells. n=5. *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference.

Fig 4: Sorafenib decreases inflammatory Th1 cells in the pancreas of prediabetic NOD mice. Flow cytometry analysis of the frequency and number (A–D) of Th1, Th2, Th17 cells and Tregs in pancreas from vehicle-treated or sorafenib-treated prediabetic NOD mice. Flow cytometry analysis of the frequency and number (E, F) of Tc1 and Tc2 cells in pancreas from vehicle-treated or sorafenib-treated prediabetic NOD mice. (G) RT-PCR analysis of transcription factors (Foxp3, Tbet, Gata3, Rorc and Bcl-6) and cytokines (Ifn-γ, Il-1β, Il-6, Il-21 and Tnf-α) in pancreas from vehicle-treated or sorafenib-treated prediabetic NOD mice. Sora, sorafenib. Data represent means ± s.e.m. and data were pooled from 2 or more independent experiments. n = 5-7 mice per group in each experiment. Statistical significance: *P < 0.05 versus vehicle by t-test.

Fig 5: In vitro and in vivo generation of antigen-specific Tregs by nanoparticles.(A) Proportion of OVA-specific Tregs in iLN following intradermal injection of different types of nanoparticles to OT-II transgenic mice that have T cells specific for OVA only, as evaluated by flow cytometry (n = 4). (B) Proportion of Tregs generated in vitro from OT-II CD4+ T cells following coculture of the CD4+ T cells with DCs that had been treated with various types of nanoparticles in vitro (n = 5). (C) Proportion of Tregs in splenocytes. The splenocytes were isolated from wild-type OA mice that had been treated with intradermal injection of different types of nanoparticles and subsequently restimulated in vitro with Col II or OVA (n = 5). (D) Relative mRNA expression of FOXP3 and TGF-β in the splenocytes restimulated in vitro with Col II or OVA (n = 6). (E) Proportion of Tregs in splenocytes analyzed by flow cytometry. The splenocytes were isolated from wild-type OA mice that had been treated with intradermal injection of different types of nanoparticles and subsequently restimulated in vitro with Col II259–273 (epitope) or OVA (n = 5). (A to E) *P < 0.05, **P < 0.01, and ***P < 0.001. One-way ANOVA (multiple comparison) with Tukey’s post hoc test.