Fig 2: 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-1ß, IL-6, and TNF-a expression in colitic tissue (n = 10–15).(E) qRT-PCR of Smad7, TGF-ß, 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-?, IL-17a, and PD1 expression in CD4+ MLN T cells (n = 5–10).(H) Representative FACS plots (left) and frequencies (right) of Foxp3+, IFN-?+, 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 3: Absence of Opg ameliorates DSS-induced colitis.a Daily changes in body weight during dextran sodium sulfate (DSS)-induced colitis. Changes in body weight percentage were calculated by dividing the body weight on the specified day by the body weight at day 0. b Colon length was measured after sacrifice at day 8. c Representative histology of hematoxylin- and eosin-stained colonic tissue from WT and Opg-/- mice with DSS-induced colitis on day 8. d Stool scores were measured as described in the Methods. e Spleen weight was measured after sacrifice at day 8. a, b, d, and e Data are presented as the mean ± SEM. **p < 0.01, ***p < 0.005 (Student’s t-test, n = 5 animals). Similar results were obtained from three independent experiments, and representative data are shown. f The number of CD45+CD3e-B220- cells and the frequency of monocytes, Ly6C+ macrophages, Ly6C- macrophages, CD103+ dendritic cells (DCs), and neutrophils in the population of CD45+CD3e-B220- cells of the colonic lamina propria were quantified by flow cytometry. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *p < 0.05; Student’s t-test, n = 5 animals. The source data underlying panels a, b, d, e and f are provided as a Source Data file.

Fig 4: ROCK blockade increases DC-mediated T cell priming. a The percentage of CD40+ and CD86+ DCs in tumour-draining LNs from B16F10-Ova tumour-bearing mice was determined by flow cytometry (n = 3). b, c B16F10-Ova tumour-bearing mice were injected (i.v.) with Y27632 or vehicle. On day 20, mice were injected (i.v.) with CFSE-labelled OT-I T cells; after 3 days, the proliferation of OVA-specific OT-I T cells in tumour-draining LNs was analysed by flow cytometry. b Representative flow cytometry plots. c Percentages of the indicated CFSE-labelled OT-I T cells (n = 4). d, e CD11c+ DCs or F4/80+ macrophages from B16F10-Ova tumour-bearing mice treated with Y27632 or vehicle were magnetically isolated from tumour-draining LNs (d) or tumours (e). DCs or macrophages were incubated with OT-I T cells for 72 h, and secreted IFN-? was analysed by ELISA (n = 3). f CD11c+ DCs or F4/80+ macrophages were isolated from tumour-draining LNs from B16F10-Ova tumour-bearing mice treated with Y27632 or vehicle and co-cultured with OT-I T cells for 48 h. The percentage of IFN-?-producing CD8+ T cells was analysed by flow cytometry (n = 3). g CD11c+ DCs from B16F10-Ova tumour-bearing mice treated with Y27632 or vehicle were magnetically isolated from tumour-draining LNs or tumours. The percentages of CD11b+, CD103+ and CD8+ DCs were analysed by flow cytometry (n = 3–6). h In CD11b+, CD103+ and CD8+ DCs from tumour-draining LNs or tumours, the percentage of H-2kb-OVA+ DCs was analysed by flow cytometry (n = 3–6). i CD11c+ DCs were isolated from tumours in B16F10-Ova tumour-bearing BALB/c mice injected (i.v.) with Y27632 (10 mg kg-1) and incubated with CMFDA-stained B16F10-Ova for 2 h, and the percentage of CD11b+ or CD103+ DCs harbouring tumour cells was determined by flow cytometry (n = 3). j CD11c+ DCs were isolated among tumour-draining LNs from B16F10-Ova tumour-bearing BALB/c mice injected (i.v.) with Y27632 (10 mg kg-1), and the percentage and relative MFI of CD40+ cells in CD103+ or CD8+ DCs was analysed by flow cytometry (n = 3). Data are presented as means ± s.d. (*P < 0.05, **P < 0.01, ns, not significant; significance determined by unpaired Student’s 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.