Supplementary Figure 3: Characterization of mouse T_RM cells and comparable abundance of CD4⁺ Hobit–Blimp-1 double-knockout (DKO) and WT T cells and T_RM cells in transfer colitis.

(a) Representative gating strategy for mouse CD4⁺ CD44⁺CD69⁺ T_RM cells. From left to right: After exclusion of doublets and dead cells, lymphocytes were selected in the foreward-/sideward-scatter. CD3⁺CD4⁺ T cells were identified and CD44 was plotted against CD69 to select CD44⁺CD69⁺ T_RM cells. (b) Flow cytometric quantification of CD44⁺CD69⁺CD103⁺ T_RM frequency in LPMCs of Rag1^-/- mice after transfer of naïve CD4⁺ T cells. Left panels: Representative dot plots of CD3⁺CD4⁺CD44⁺ LPMCs one week and six weeks after transfer; the frequency of CD69⁺CD103⁺ T_RM cells among lymphoid LPMCs is indicated. Right panels: Quantification of CD103⁺ CD4⁺ T_RM cell frequency over time. n = 5 (1+2 weeks), n = 4 (3+4 weeks), n = 3 (5 weeks). (c) Flow cytometry of the expression of the T_RM-associated molecule CD49a on CD44⁺CD69⁺ T_RM cells from the intestinal lamina propria (LP) and splenic CD44⁺ memory T cells. Histograms representative for two independent experiments are shown. (d) Immunohistochemistry staining for CD4 in colon tissue of Rag1^-/- mice after transfer of naïve CD4⁺ WT, Hobit KO or DKO cells. Left panels: Representative images. Right panel: Quantitative data. Bars: 50 µm (upper row), 25 µm (lower row). n = 19 from two independent experiments. (e) Left: Competitive in vivo homing assay (n = 8) with CD4⁺ T cells from DKO (green) and WT (magenta) mice transferred to the ileocolic artery of Rag1^-/- mice. Representative intravital confocal microscopy with arrows highlighting extravasated cells and quantification of homed cells per region of interest (ROI). Right: Non-competitive in vivo homing assay (n = 10) with CD4⁺ T cells from DKO and WT mice transferred to the ileocolic artery of Rag1^-/- mice. Representative dot plots of homed CFSE⁺ cells in the LP and quantification. (f) Flow cytometry assessment of proliferation and apoptosis of anti-CD3/CD28-stimulated CD4⁺ WT and DKO T cells. Left panels: Representative flow cytometry showing CFSE dilution after culture of CD4⁺ T cells for 48 hours. Right panels: Quantification of proliferation and apoptosis as indicated. n = 10. (g) Flow cytometry of CD69 and CD103 expression in CD4⁺ LPMCs from Rag1^-/- mice after transfer of WT or DKO cells. Representative histograms and quantitative data from one representative experiment with six mice out of two independent experiments are shown. (b, d) Center values – mean; error bars – s.e.m., (e,f) Boxplots – median with upper and lower quartile, whiskers – minimum and maximum. (g) Floating bars - median with minimum and maximum. (d) One-way ANOVA, (e-g) two-tailed student’s t-test. n.s. – not significant.