Fig 2: IL-17A-Producing Blimp1 −/− Foxp3+ Treg Have Impaired Suppressive Function and Can Cause Intestinal Inflammation(A) Body weight of RAG−/− mice injected with naive CD45.1+CD4+T cells alone or in combination with (CD45.2+) Blimp-1 −/−IL-17A-producing (IL17AGFP+ Treg) or non-producing (IL-17AGFP− Treg) Foxp3+ Treg from the same donor mice.(B and C) Colon length (B) and H&E-stained histological sections (C) (10x magnification; scale bar, 100 μm) in mice shown in (A) 8 weeks after transfer.(D) Frequency (fluorescence-activated cell sorting [FACS] plots) and absolute numbers (bar graphs) of IL-17AGFP+ cells from LI-LP of mice shown in (A)-(C).(E) Numbers of CD45.1 + cells in the LI-LP, MLNs, and SP from mice shown in (A)–(D).(F) IL-17A expression (ELISA) in LI-LP cells of mice shown in (A)ߝ(E).(G) Body weight of RAG−/− mice adoptively transferred with IL-17+ Th17 cells (Th17), Blimp1 −/− IL17AGFP+, or IL17AGFP−Foxp3+ Treg.(H) Histology scores (colon, rectum, and cecum; left), and pictures of colon histological sections (H&E stained; 10x magnification; scale bar, 100 μm) of mice shown in (G).(I) IL-17A expression in TCRβ+ T cells from the LI-LP of mice show in (G). Foxp3+, cells that maintained Foxp3; Foxp3−, cells that had lost Foxp3 expression after transfer.(J) Expression of IL-17A and IL-17F in TCRβ+Foxp3+ and Foxp3− cells in SP of mice shown in (G)–(I).Data shown are from three different experiments. Error bars show SEM (n = 3–7 mice per group). *p < 0.05 and **p < 0.01, one-way ANOVA (A, B, D, and F–I) and unpaired Student’s t test (E and I).

Fig 3: Increased Binding of IRF4 at the Il17 Locus and IRF4 and RORγt Requirement for IL-17A Production in Blimp-1 −/−Foxp3+ Treg(A) Representation of the Il17 locus showing RORγt and IRF4 consensus binding sites (top) and RORgt ChIP (bottom) in Blimp-1+/+ and Blimp-1 −/−CD4+ Foxp3GFP+ Treg and wild-type pTh17 cells.(B) IL-17A expression in TCRβ+CD4+Foxp3+ Treg (MLNs) from Prdm1+/+Rorc+/+Foxp3CREYFP+ (Ctrl), Prdm1F/FRorc+/+Foxp3CREYFP+ (knockout [KO]), and Prdm1 F/FRorcF/FFoxp3CREYFP+ (double knockout [DKO]) mice and expression of Foxp3, Prdm1, Rorc, and Il17a mRNA (bottom graphs) in sorted and in vitro-stimulated (phorbol myristate acetate [PMA] and ionomycin for 4 hr) CD4+Foxp3CREYFP+ cells.(C) IRF4 ChIP at the Il17 CNS7 and Il10 Intron I regions in Blimp1+/+ and Blimp1 −/−CD4+Foxp3GFP+ Treg and wild-type pTh17 cells.(D) Irf4, Il17a, and Il10 mRNA expression following Irf4 siRNA knockdown (SC, scrambled control).Data shown are representative of at least two independent experiments. Error bars show SEM (n ≥ 3 per group). *p < 0.05 and **p < 0.01, one-way ANOVA.

Fig 4: FACS analysis of PPs and spleen samples between two groups after FMT from RA patients or healthy controls.A Representative images of different expression of CD4+ T cells (% of total cells) in PPs and spleen. PPs (Student t-test: t = −4.944, P < 0.001); spleen (Student t-test: t = −2.258, P = 0.036). B Representative images of different expression of Treg cells (Foxp3+CD25+, % of CD4+ T cell) in PPs and spleen. PPs (Student t-test: t = 7.443, P < 0.001); spleen (Student t-test: t = 4.290, P < 0.001). C Representative images of different expression of Th1 cells (IFN-γ+, % of CD4+ T cell) / Th2 cells (IL-4+, % of CD4+ T cell) in PPs and spleen. PPs (Student t-test: t = −2.860, P = 0.010); spleen (Student t-test: t = −2.955, P = 0.008). D Representative images of different expression of Th17 cells (IL-17A+, % of CD4+ T cell). PPs (Student t-test: t = −0.042, P = 0.967); spleen (Student t-test: t = 0.426, P = 0.675).

Fig 5: Blockade of Tim-3 exacerbated multiple sclerosis in the EAE model. MOG was emulsified into CFA, mice were immunized subcutaneously, and pertussis toxin (500 ng/mouse) was injected intraperitoneally at 2 and 48 h after immunization. The immunized WT mice were divided into two groups (n = 10): one group is injected with anti-Tim-3 antibody, and the other group is injected with isotype antibody (10 mg/kg, intraperitoneal injection every other day. On the 20th day after immunization, the mouse spinal cords, peritoneal macrophages, and spleens were harvested. (A) Flow cytometry was used to detect the expression of MHC-II on the surface of macrophages. The percentage of MHC-II and the GeoMean were counted (mean ± SD, n = 5), (B) and the expression level of MHC-II was detected by RT-PCR (mean ± SD, n = 6) and Western blotting. (C) Flow cytometry was used to detect the activation levels of IL-17+CD4+T, IFN-γ+CD4+T, and Foxp3+CD4+T cells in splenic lymphocytes (mean ± SD, n = 6–10). (D) The weight and clinical scores of the EAE mice were recorded from the 10th day. (E) The mouse spinal cord tissue was isolated and stained with HE and LFB (*p < 0.05; ****p < 0.0001).