Fig 2: Strong memory CD4+ T cell recall following intravaginal Ag exposure in Ag–IRES–CCL25 immunized mice. Flow cytometry analysis of lymphocytes isolated from spleens (A), small intestine (B), and vaginal mucosa (C) of naive (black bars), Ag–IRES–scb (gray bars), Ag–IRES–CCL20 (blue bars), and Ag–IRES–CCL25 (purple bars) and stained with mAb panel 2 (cf. Table II). The gating strategies are found in Supplemental Figs. 3D, 4A, and 4B. The different T cell subpopulations are reported as percent of CD4+ or CD8+ T cells with naive T CD44low CD62Lhigh, Tmem CD44high CD62Llow, and central memory T CD44high CD62Lhigh. The panels on the right depict the surface expression of CD69/CD103 as percentage of CD4+ Tmem in the spleen (D) and in the vaginal mucosa (F) as well as in the intestinal mucosa for CD8+ Tmem (E). For panels on the left, error bars represent mean ± SEM, and for panels on the right, data points are shown as box and whiskers, min/max, with n = 8 animals per group and n = 7 for Ag–IRES–scb and Ag–IRES–CCL25. Two-way ANOVA with Dunnett correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. See also Supplemental Fig. 4C–E.

Fig 3: Smad7 Deficiency Promotes a PDL2+CD103+ Regulatory Phenotype in DCs(A) Representative fluorescence-activated cell sorting (FACS) histograms (left) and median fluorescence intensities (MFIs) (right) of PDL2, PDL1, and CD103 in CD11c+ MLN DCs from naive Smad7fl/fl and DC-Smad7−/− mice (n = 11).(B) ELISA quantification of secreted TGF-β by MLN DCs from these mice (n = 4).(C) Representative FACS histograms (left) and MFIs (right) of PDL2, PDL1, and CD103 in CD11c+ BMDCs generated with or without TGF-β (30 ng/mL) from WT mice (n = 5).(D) Western blot of Smad7 in WT CD11c+ BMDCs stimulated with or without TGF-β (2.5 ng/mL) for 30 min.(E) ChIP analysis of Smad3 binding promoter regions of Pdcdl2 (PDL2), Pdcdl1 (PDL1), and Itgae (CD103) in WT CD11c+ DCs stimulated with or without TGF-β (10 ng/mL). ChIP data expressed as fold enrichment as compared to input control (n = 3).(F and G) Luciferase reporter activity of Pdcdl2, Pdcdl1, and Itgae promoters in HEK293 cells stimulated (F) with control or Smad3 construct or (G) with or without TGF-β (2 ng/mL) (n = 3). Luciferase activity as relative activity fold change (FC) from (F) control or (G) media condition.(H) Representative FACS histograms (left) and MFIs (right) of PDL2 and PDL1 in CD103+ and CD103− MLN CD11c+ DCs from WT mice (n = 4).Data representative of ≥3 independent experiments. MFI data reflective of CD11c+ population and expressed as FC from (A) Smad7fl/fl condition, (C) media condition, or (H) CD103− condition. Means ± SEMs. *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired t test. See also Figure S1.

Fig 4: Smad7 Inhibition Ameliorates DSS-Induced Colitis in Mice(A) Schematic of DSS colitis induction (3% DSS in water for 7 days, untreated drinking water for 7 days) and Smad7-as treatment i.p. (250 μg per mouse every other day 4 times).(B) Percentage body weight changes (left) and linear regression analysis (right) of colitic mice treated with Smad7-as or its oligonucleotide control (n = 8).(C) Representative histopathological sections with H&E (left) and scores (n = 8) (right) based on the degree of ulceration at day 14, scored blinded by a pathologist at HRHCF. Scale bars represent ~1 mm (top) and ~100 μm (bottom).(D) qRT-PCR of Smad7, IL-1β, IL-6, and TNF-α expression in colitic tissue (n = 10–15).(E) qRT-PCR of Smad7, TGF-β, PDL2, PDL1, and CD103 expression in MLN CD11c+ DCs from colitic mice (n = 10).(F) Representative FACS histograms (left) and MFIs (right) of PDL2, PDL1, and CD103 in MLN (top) and splenic (bottom) CD11c+ DCs from colitic mice (n = 5–10). MFI data reflective of CD11c+ population and expressed as FC from control condition.(G) qRT-PCR of Smad7, Foxp3, IFN-γ, IL-17a, and PD1 expression in CD4+ MLN T cells (n = 5–10).(H) Representative FACS plots (left) and frequencies (right) of Foxp3+, IFN-γ+, and IL-17a+ populations in CD4+ MLN (top), splenic (center), and LP (bottom) T cells (n = 4–5).(I–K) DSS colitis was induced (3% DSS in water for 7 days, untreated drinking water for 7 days) and PDL1-fc or PDL2-fc treatment were given i.p. (100 μg per mouse every other day 4 times) as in (A). (I) Percentage body weight changes (left) and linear regression analysis (right) of colitic mice treated with PDL1-Fc, PDL2-Fc, or Ig control (n = 5–7). (J) Representative histopathological sections (left) and scores (right) based on the degree of ulceration, scored blinded by a pathologist at HRHCF (n = 8). Scale bars represent ~1 mm (top) and ~100 μm (bottom). (K) qRT-PCR of Foxp3 expression in colitic tissue (n = 4–5).Data representative of ≥3 independent experiments. qRT-PCR and MFI data expressed as FC from control condition. Means ± SEMs. *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired t test.

Fig 5: 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.