WT1 specific T cells display peptide-specific effector function. (A) In vivo cytotoxicity of CFSE-labeled peptide-loaded target cells. A2Kb Tg mice, 11 weeks after transfer of BM stem cells transduced with the WT1-TCR (top panel, n = 7 A2Kb → A2Kb, n = 5 B6 → A2Kb) or LMP2-TCR (bottom panel, n = 4 A2Kb → A2Kb) were intravenously injected with a 1:1 mix of relevant: irrelevant peptide-loaded A2Kb Tg splenocytes, differentially labeled with CFSE (WT1-TCR is specific for WT126 peptide, and LMP2-TCR is specific for CLG peptide). Eighteen hours later, splenocytes of injected animals were harvested and analyzed by FACS to identify CFSE-labeled cells. Representative plots are shown. Control untreated A2Kb Tg mice were injected with CFSE-labeled peptide-loaded target cells. Summary data of in vivo cytotoxicity assays are shown on the right. Percentage antigen-specific cytototoxicity was calculated as described in “In vivo cytotoxicity assays.” ***P < .001. (B) Ex vivo proliferation of splenocytes from mice previously transplanted with WT1-TCR and LMP2-TCR-transduced A2Kb or B6 BM stem cells. Splenocytes were stimulated for 5 days with 100μM relevant or irrelevant peptide. CFSE-labeled splenocytes were analyzed by FACS for CFSE dilution after anti–human Vβ2.1 or Vβ13 antibody staining. A representative plot of pWT126-specific proliferation is shown on the left. Summary data of ex vivo proliferation assays using T cells harvested from mice transplanted with WT1-TCR (n = 8 A2Kb → A2Kb, n = 3 B6 → A2Kb) and LMP2-TCR (n = 5 A2Kb → A2Kb) transduced BM stem cells is shown on the right. **P < .01, ***P < .001. (C) Splenocytes harvested from A2Kb mice transplanted with WT1-TCR and LMP2-TCR-transduced A2Kb BM stem cells were stimulated ex vivo with 100μM relevant or irrelevant peptide for 5 days. ELISAs were performed to detect antigen-specific IFN-γ and IL-2 secretion. ns indicates not significant.