Fig. 5. TGF-β signals via Smad3/4 to regulate IL-10 in T_H17 cells.

a Flow cytometric analysis of small intestinal CD4⁺ T cells after anti-CD3 mAb. The indicated reporter mouse lines were used. Top panels are pre-gated on Foxp3⁻, CD4⁺ T cells. Bottom panels are pre-gated on YFP⁺ cells, and the indicated populations are identified on the basis of the reporter molecules as follows: T_R1^exTH17 cells: IL-10^eGFP+ IL-17a^Kata−; IL-10⁺ T_H17 cells: IL-10^eGFP+ IL-17a^Kata+; T_H17 cells: IL-10^eGFP− IL-17a^Kata+. b, c Frequencies (b) and numbers (c) of T_R1^exTH17, IL-10⁺ T_H17 and T_H17 cells in A are one representative experiment out of three. Each dot represents one mouse (n_{wild type} = 5, n_KO = 5). Mean ± S.D.; ns, not significant; *P < 0.05, **P < 0.01 by Mann-–Whitney U test. d Experimental design of knocking out Smad3 in in vitro differentiated mature T_H17 cells by using CRISPR/Cas9 technology. e Flow cytometric analysis of in vitro cultured mature T_H17 cells after knocking out Smad3 by using gRNAs targeting Smad3. Cells were pre-gated on viable CD4⁺ T cells. NT, non-targeting control. Each dot represents an individual gRNA (n_NT = 2, n_Smad3 = 3). Mean ± S.D.; **P < 0.01 by Welch’s t-test. Source data are provided as a Source data file.