TGF-β downregulates ZEB2 and enforces the retention of T_RM cell precursors in the skin. (A) Naive P14 CD45.1⁺ T cells were transferred into recipient mice infected with LCMV. P14 T cells were isolated from the spleen 7 dpi and cultured with TGF-β in vitro for 2 d. Expression of the indicated genes was determined by qPCR and represented as a heatmap (z-score normalized by row). (B) Effector CD45.1⁺CD45.2⁺ OT-I (OT-I WT) and CD45.1⁺ OT-I Tgfbr2^−/− (OT-I Tgfbr2^−/−) were transferred into mice infected with HSV-OVA. OT-I T cells from the skin were isolated 14 dpi, and expression of the indicated genes determined by RNA sequencing and represented as a heatmap (z-score normalized by row). (C) Naive gBT-I T cells were nucleofected with control-nontargeting (GFP⁺ gBT-I sgCtrl) or Zeb2-targeting (CD45.1⁺ gBT-I sgZeb2) sgRNA/Cas9 RNPs and cotransferred into WT mice. Recipient mice were infected with HSV, and relative frequencies of gBT-I T cells were quantified in the spleen and skin (total gBT-I or CD69⁺CD103⁺ gBT-I T_RM) 14 dpi. Data are representative of two independent experiments, with n = 7–9 mice per experiment. (D and E) Naive OT-I T cells sufficient (OT-I WT) or deficient in S1PR5 (OT-I S1pr5^−/−) were transferred at a 1:1 ratio into recipient mice that were subsequently infected with HSV-OVA. Shown are relative frequencies of WT and S1pr5^−/− OT-I T cells isolated from the spleen and skin (total OT-I or CD69⁺CD103⁺ OT-I T_RM) at 8, 14, and 30 dpi. In C, data are representative of two independent experiments, with n = 7–9 mice per experiment. In D and E, data are representative of four independent experiments with n = 9–10 mice per group per experiment. Unpaired t test was used in C and two-way ANOVA in E. ****, P < 0.0001. freq, frequency; n.s., not significant; T_N, naive T cell.