Defective stem-like CD8 ⁺ T cell maintenance in the absence of TGF-β signaling. Same experimental setup as in Fig. 1. (A) At indicated days after infection, the expression of CXCR5 on splenic P14 T cells was determined. (B) The percentage of CXCR5⁺ cells in donor P14 T cells is shown. Upper, spleen; lower, lymph nodes (LNs). Representative (A) or pooled (B) results from four independent experiments are shown (n = 4–14/time point). (C) Day 15–25 after infection, pre-gated Tcf-1⁺ and Tcf-1⁻ WT splenic P14 T cells were examined by flow cytometry. Representative FACS profiles from three independent experiments (n = 5/each) are shown. (D–F) Representative FACS profiles of pre-gated donor WT and Tgfbr2^−/− P14 T cells are shown (three repeats, n = 10). Representative results from three independent experiments are shown. (G) Phosphorylated Smad2/3 was measured by FACS. Each symbol represents the results from an individual mouse. Pooled results from two independent experiments are shown in G (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 by paired Student’s t test (B) or one-way ANOVA (G).