F5 effector cells develop into functional long-term resting memory in the absence of IL-7R expression in Rag1^–/– hosts. Splenocytes from F5 control mice and F5 TreIL-7R donors taken off dox food 3 days previously were stimulated with NP68 peptide for 72 hours followed by a further 96 hours of culture in IL-2. Blasts from cultures of F5 TreIL-7R^OFF and control F5 T cells were mixed in equal numbers and 2 × 10⁷ total cells transferred into Rag1^–/– and Il7^–/–Rag1^–/– recipients (n = 6). Recipient mice were bled at different times after transfer and analyzed for CD8, TCR, and Ly5.1 expression. (A) The graph shows the frequency of Ly5.1-negative Ly5.2-positive F5 TreIL-7R^OFF T cells (○) and Ly5.1-positive F5 control cells (•) in PBLs of Rag1^–/– hosts. (B) The graph shows the proportion of donor CD8⁺ T cells that were of Ly5.1-negative Ly5.2-positive F5 TreIL-7R^OFF origin in Rag1^–/– (•) and Il7^–/–Rag1^–/– (○) hosts. (C) Antigen-specific in vivo killing function was determined in Rag1^–/– recipients of either 2 × 10⁷ F5 TreIL-7R^OFF or 4 × 10⁶ control F5 cultured effector cells 7 weeks after transfer. Targets were prepared and injected into hosts as described in Figure 3C and killing determined by comparison with targets transferred to empty Rag1^–/– hosts 24 hours later. (D) Dot plots show FSc versus SSc profiles of the indicated F5 blasts after 7 days of culture in vitro and 1 day after transfer in vivo. (E) Histograms show CD122 expression by F5 TreIL-7R^OFF (solid line) and F5 control T cells (gray fill) ex vivo and at day 7 culture in vitro. Reference histogram gates are identical in width and position with respect to CD122 staining as circular gates shown in panel F set by gating CD122^hiCD44^hiCD8⁺ memory phenotype cells in WT C57BL/6J mice. (F) Dot plots show CD44 versus CD122 expression by F5 TreIL-7R^OFF blasts and F5 control blasts in peripheral blood 1 day after transfer and plots of peripheral blood from WT C57BL/6J and naive F5 Rag1^–/– controls. Data are representative of 4 or more experiments.