Fig 2: Srsf1-deficient Treg cells exhibit impaired phenotype and suppressive function(A) Spleen cells were isolated from WT and T cell Srsf1-KO mice, and expression levels of Treg-associated molecules in CD4+CD25+Foxp3+ Treg cells were analyzed by flow cytometry. Dot plots (left) and histograms (right) show representative data of frequencies and mean fluorescence intensity (MFI) of markers in gated Treg cells.(B) Graphs show the percentage of CTLA4+, PD1+, and CD39+ cells gated on Treg cells and quantification of MFI of CD103, ICOS, GITR, LAG3, and Helios (n = 4–10 each).(C) Spleen cells from WT or T cell Srsf1-KO mice were isolated and conventional CD4 T (Tconv) cells and Treg cells (CD4+CD25+CD127lo) were sorted by flow cytometry. Tconv cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with Treg cells at increasing ratios for 7 days, and proliferation of Tconv cells was analyzed by flow cytometry. Representative plots are shown.(D) Graph shows proliferation of Tconv cells (n = 8 WT, n = 10 KO).(E) B6 mice were given 2.5% dextran sodium sulfate (DSS) in water to induce colitis for 8–10 days. One day before the initiation of DSS administration, PBS or flow-cytometry-sorted Treg cells (CD4+CD25+CD127lo) from WT or T cell Srsf1-KO mice were injected into B6 mice. Graph shows body weight of mice from five independent experiments (n = 6 each).(F) Representative image of colon after DSS administration for 8–10 days. Graph shows colon length of WT Treg and KO Treg group (n = 6 WT, n = 5 KO).(G) Naive CD4 T cells were adoptive transferred into Rag1-/- mice to induce colitis together with PBS or sorted Treg cells (CD4+CD25+CD127lo) from WT or T cell Srsf1-KO mice. Graph shows time course of body weight of Rag1-/- recipient mice (n = 4–5 each).*p < 0.05; **p < 0.005; and n.s., no significant difference, two-way ANOVA test (D, E, and G) and unpaired t test (B and F); mean ± SEM.

Fig 3: Schematic of cellular and molecular mechanisms through which Nox2 in CD4+ T cells regulates Ang II–induced hypertension and heart remodeling.Deficiency of Nox2 in Tregs results in increased numbers of Tregs in the heart and vessels and protects against the development of hypertension, interstitial cardiac fibrosis, and cardiomyocyte hypertrophy. Mechanistically, the deficiency of Nox2 in Tregs enhances their suppressive function through an increase in nuclear levels of FoxP3 and NF-?B activation, and higher levels of GITR, CTLA-4, CD39, CD73, and CD25. The higher CD25 levels may drive an increased STAT5 phosphorylation under IL-2 stimulation, and lead to a positive feedback promoting suppression. The deficiency of Nox2 also drives a change in balance between Tregs and Th17 cells to a more antiinflammatory profile, with higher IL-10 and lower IL-17 levels.

Fig 4: CD39+?d Tregs derived from RSCRC showed enhanced immunoregulatory function compared with LSCRC.(A) HPLC assay to measure adenosine concentrations in the supernatants of CD39+?d Tregs on day 5. Data are shown as mean ± SEM, using 2-way ANOVA followed by Sidak’s multiple-comparisons test; n = 5/group. Sorted CD39+?d Tregs from RSCRC and LSCRC were in vitro cocultured with CFSE-labeled allogeneic CD4+/CD8+ T cells in the presence of CD3 and CD28 mAbs. (B and D) Representative histogram plot of CD8+ T cells and CD4+ T cells. (C and E) Bar diagram summarizes the percentages of proliferated cells (CFSElo) in CD8+ T cells (C) and CD4+ T cells (E). (F and H) Representative flow cytometric analysis of IFN-? in CD8+ T cells (F) and CD4+ T cells (H). (G and I) The percentages of IFN-?+ cells in CD8+ T cells (G) and CD4+ T cells (I) was summarized. One-way ANOVA followed by Tukey’s multiple-comparison test was used for multiple-group analysis. Data are shown as mean ± SEM. RSCRC, n = 3; LSCRC, n = 3 from 3–5 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: The infiltration of CD39+?d Tregs was significantly increased in RSCRC compared with LSCRC.(A) Representative flow cytometric analysis of the distribution of CD39+ Tregs in CRC and paired normal tissues. (B–D) The percentages of CD39+ ?d Tregs (B), CD4+ T cells (C), and CD8+ T cells (D) in the total tumor-infiltrating CD3+ T cells was calculated. Data are shown as mean ± SEM, using 2-way ANOVA followed by Sidak’s multiple-comparisons test. RSCRC, n = 15; LSCRC, n = 20. Each dot represents 1 patient. N, normal; T, tumor; RSCRC, right-sided colorectal cancer; LSCRC, left-sided colorectal cancer. **P < 0.01, ***P < 0.001.