NKG2D is expressed on antigen-experienced CD4⁺ T cells in steady-state C57BL/6 mice. (A) Representative FC analysis of NKG2D expression on single-cell suspensions from the spleen of C57BL/6 mice. (B) Representative FC analysis of CD44 and CD62L expression on NKG2D⁺ and NKG2D⁻ CD4⁺ T cells in the spleen. (C) Frequency of CD44⁺ CD62L⁻ T cells; quantification from B; n = 8; Wilcoxon test. (D) Representative FC analysis of NKG2D expression on CD44⁺ CD4⁺ T cell from single-cell suspensions of C57BL/6 mice. (E) Frequency of NKG2D⁺ CD44⁺ CD4⁺ T cells in indicated organs, spleen (n = 29), BM (n = 15), siLP (n = 15), colon lamina propria (cLP; n = 7), liver (n = 6), and lung (n = 7). (F and K) Representative FC analysis of CXCR3 (F) and CCR6 (K) expression on CD4⁺ CD44⁺ NKG2D⁻ (black line) or NKG2D⁺ (red line) in spleen and siLP, respectively. Shaded histogram represents live CD19⁺ (F) or live CD8⁺ (K) cells; n = 6. (G and L) Representative FC analysis of T-bet expression in spleen (G) and RORγt and T-bet expression in siLP (L) in CD4⁺ CD44⁺ NKG2D⁻ or NKG2D⁺ T cells. (H and M) Frequency of T-bet⁺ CD44⁺ CD4⁺ T cells in spleen (H), quantification from G (n = 11) and T-bet⁺ or RORγt⁺ CD44⁺ CD4⁺ T cells in siLP (M), quantification from L (n = 12); Wilcoxon test. ns, not significant. (I) Representative FC analysis of IL18R1 and ZsGreen (Tbx21) expression on CD4⁺ CD44⁺ NKG2D⁻ and CD4⁺ CD44⁺ NKG2D⁺ in spleen of Tbx21-ZsGreen mice (n = 3). (J) Frequency of splenic NKG2D⁺ CD44⁺ CD4⁺ T cells from WT (n = 15) and Tbx21^−/− mice (n = 13); Mann–Whitney U test. In C, E, H, J, and M, each symbol represents a mouse, lines represent mean ± SEM, and data are pooled from at least three independent experiments.