WT1-specific memory phenotype T cells display antigen specific effector function. (A) T cells were purified from A2Kb mice transplanted with WT1-TCR-transduced B6 BM stem cells and used for adoptive transfer into irradiated secondary A2Kb Tg recipient mice (n = 4). At 10 weeks after transfer, CD8/TCR expression of the transferred T cells was determined by FACS analysis of splenocytes, LNs, and BM after staining with antimurine CD8 and antihuman Vβ2.1 antibodies, respectively. (Left panel) The TCR and CD8 profile of the T cells before transfer. (Right panel) The profile 10 weeks after transfer. Percentage of TCR^hi and TCR^lo populations are shown after gating on total CD3⁺ cells. (B) Analysis of the naive/memory phenotype of gated WT1-TCR⁺ (Vβ2.1⁺) T cells from spleen, LNs, and BM 10 weeks after adoptive transfer was determined by FACS analysis after staining with anti-CD44 and CD62L antibodies. (Left panel) The naive/memory phenotype of gated WT1-TCR⁻ T cells in LNs. (C) In vivo cytotoxicity experiments were performed in A2Kb Tg secondary recipients 10 weeks after adoptive transfer of T cells. Mean percentage killing of CFSE-labeled relevant peptide-loaded splenocytes from 3 mice is shown on the left. Ex vivo proliferation of WT1-TCR⁺ and WT1-TCR⁻ T cells after stimulation with relevant peptide was determined by FACS analysis for CFSE dilution after staining with antihuman Vβ2.1 antibodies.