CD8 T-cell tolerance is lost after transfer into irradiated lymphopenic mice dependent on the presence of IL-15. (A) Splenocytes (2 × 10⁶) from thymectomized Des-TCR × 2.4KerIV-K^b.RAG2^−/− mice (tolerant) and thymectomized Des-TCR.RAG2^−/− mice (reactive) were injected intravenously into CBA mice irradiated with a dose of 6 Gy 1 day before transfer. Five days after transfer, recipient splenocytes were stimulated with anti-CD3 and analyzed by flow cytometry. Shown is the mean percentage of IFN-γ–producing CD8 T cells ± SD after gating on Des-TCR⁺ CD8 cells. Results are from 3 independent experiments which were summarized (n = 8). (B) Splenocytes (2 × 10⁶) from thymectomized Des-TCR × 2.4KerIV-K^b.RAG2^−/− mice (tolerant) were injected intravenously into RAG2^−/− mice irradiated with a dose of 3 Gy 24 hours earlier. Nonirradiated control mice served as controls. P815.K^b.B7 cells were injected subcutaneously 3 days after T-cell transfer. The mice were monitored for up to 4 weeks for the occurrence of tumor growth. Data represent the respective percentage of tumor-bearing mice pooled from 3 independent experiments (n = 10). Alternatively, recipient RA2^−/− mice were treated with melphalan (7.5 mg/kg intraperitoneally) 2 days before T-cell transfer, with IL-15/IL-15Rα complexes (1.5 μg of IL-15 and 7 μg of sIL-15Rα-Fc intraperitoneally 1 day after injection of tumor cells) or received P815.K^b.B7–IL-15 tumor cells (n = 7 for each group). (C) CSFE-labeled splenocytes (2 × 10⁶) from thymectomized Des-TCR × 2.4KerIV-Kb.RAG2^−/−mice (tolerant) were transferred intravenously into IL-15^−/− or littermate IL-15^+/+ mice, reisolated, and analyzed by flow cytometry (all H2-K^k background hosts were irradiated with 6 Gy 24 hours earlier, a minimum of 4 mice per time point). Shown are relative log fluorescence intensities for CFSE. (D) Similar experiments to panel A were analyzed on transfer of tolerant Des-TCR CD8 T cells into irradiated IL-15^−/− or IL-15^+/+ mice (6 Gy, H2-K^k) after 3 or 10 days (2 independent experiments and 7-9 mice per time point; **P < .01).