Fig. 3: Aberrant positioning of Helios^pos thymocytes in the absence of TGF-β signaling.

a Immunostaining on thymic sections from TGFβR-KO mice and their littermate controls (TGFβR-WT). The medulla (M) was stained with K14 (red). The cortex and the cortico-medullary junction are noted C and CMJ, respectively. The CMJ was defined using computational approaches. Foxp3^pos cells are stained in yellow and Helios^pos cells in blue. Scale bars, 50 µm  (magnication 3x). b Graph shows the density of Foxp3^neg Helios^pos cells at the CMJ. c, d Flow cytometry analysis on Foxp3^neg CD4^pos CD8^low thymocytes and Foxp3^neg CD4 SP thymocytes from TGF-βR-KO mice and TGF-βR-WT mice based on gating strategy of Fig. 1. c Countor plots illustrate the expression of CXCR3 and Helios, whereas contour plots represent the expression of BIM and CXCR3 among either Foxp3^neg Helios^pos CD4^pos CD8^low thymocytes or Foxp3^neg Helios^pos CD4 SP thymocytes. d Histograms illustrate the percentage of CXCR3^pos cells among either Foxp3^neg Helios^pos CD4^pos CD8^low thymocytes or Foxp3^neg Helios^pos CD4 SP thymocytes. Pie graphs demonstrate the expression of BIM relevant to CXCR3 expression. e, f Representative flow cytometry analysis of CXCR3 and T-Bet expression among either Foxp3^neg Helios^pos CD4^pos CD8^low thymocytes or Foxp3^neg Helios^pos CD4 SP thymocytes. Histograms illustrate the percentage of T-Bet^pos cells and CXCR3^pos cells among Foxp3^neg Helios^pos CD4^pos CD8^low thymocytes and Foxp3^neg Helios^pos CD4 SP thymocytes in CD4-cre;smad4^fl/fl (SMAD4-KO) mice, CD4-cre;trim33^fl/fl (TRIM33-KO) mice, CD4-cre;smad4^fl/fl; trim33^fl/fl (ST-KO) mice. All the experiments were conducted in 7–10-day-old animals. Data are representative of 3 experiments, with 3–4 mice per group. **P < 0.01, ***P < 0.001 (two-tailed Student’s t test). Error bar, mean ± SEM. Source data are provided as a Source Data file.