In vitro induction of anti–third-party Tcms, and their homing to recipient LNs. (A) BALB/c or CB6 splenocytes were stimulated with irradiated FVB splenocytes for 60 hours or 6 days in the absence of cytokines. Subsequently, CD8⁺ T cells were positively selected and further cultured with rhIL-2 or rhIL-15 in an Ag-free environment. Alternatively, anti–third-party CTLs were grown by culturing the cells with rhIL-2 and reactivating them with FVB splenocytes, after the 6-day cytokine deprivation period. On day 15 of the culture (end of culture), the cells were evaluated for percentage of Tcms (CD44⁺CD62L⁺) using FACS analysis. Data represent average ± SD of at least 3 independent experiments for each group. ***P < .001 compared with the cells cultured with only IL-15 after 60 hours of cytokine deprivation. (B-E) Lethally irradiated (8 Gy) BALB/c mice received 4 × 10⁶ C57BL/6-NUDE BM cells and 1 × 10⁴ syngeneic T cells. Mice then received a transplant of 1 × 10⁷ DIR-labeled, CB6-derived, purified anti–third-party Tcms (B,D) or CTLs (C,E). After 2 (B-C) or 7 (D-E) days, selected recipients were killed and images were taken ex vivo using IVIS. A representative mouse, of 6 mice in 2 independent experiments, is displayed for each group.