ZEB2 and S1PR5-deficient CD8⁺ T cells show increased T_RM cell formation in the skin. (A) Naive P14 T cells were transferred into LCMV-infected mice. P14 T cells were isolated from the spleen 7 dpi and cultured with TGF-β in vitro for 2 d. Expression of indicated genes was quantified by qPCR and normalized to a housekeeping gene. (B and C) Effector or 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 recipient mice infected with HSV. gBT-I T cells were quantified in the spleen (CD127⁺CXCR3⁺KLRG1⁻CX3CR1⁻ and CD127⁻CXCR3⁻KLRG1⁺CX3CR1⁺) 8 dpi (B) or spleen and skin (total gBT-I or CD69⁺CD103⁺ gBT-I) 14 dpi (C). (D) Naive OT-I WT and OT-I S1pr5^−/− T cells were cotransferred into recipient mice infected with HSV-OVA. Shown are numbers of WT and S1pr5^−/− OT-I T cells isolated from the spleen and skin (total OT-I or CD69⁺CD103⁺ OT-I) 14 and 30 dpi. In A, data are pooled from two independent experiments, with n = 6 mice per group. In B and C, data are representative of two independent experiments, with n = 4 mice (B) and n = 7–9 mice (C) per experiment. In D, data are representative of four independent experiments, with n = 9–10 mice per group per experiment. Mann–Whitney test was used in A, paired t test in B, and paired Wilcoxon test in C and D. *, P < 0.05; **, P < 0.01; ***, P < 0.001. A.U., arbitrary units; n.s., not significant; T_EFF, effector T cell; T_N, naive T cell.