In vitro expanded CTLs derived from NSG-HLA-A24/HHD WT1-TCR gene–transduced HSC recipients exerted HLA-restricted antigen-specific response. (A) Representative flow cytometry plots showing WT1/HLA-A*2402 tetramer analysis before and after in vitro stimulation of CTLs from BM (top), spleen (middle), and thymus (bottom) of NSG-HLA-A24/HHD recipients transplanted with WT1-TCR–transduced HSCs. The frequencies (B) and cell numbers (C) of WT1-specific tetramer⁺ CTLs derived from WT1-TCR-transduced HSC recipients before and 1 week after the second in vitro stimulation. *P < .05 and **P < .01 by ratio Student t test. (D) After expansion, recipient-derived WT1-specific CTLs exerted IFN-γ production in response to WT1 peptide–pulsed LCLs (top row), but were not responsive against peptide-unpulsed LCLs (middle row). The addition of anti-HLA class I antibody reduced the WT1 peptide–specific cytokine production (bottom row). (E) Spot counts of IFN-γ-producing cells (spot-forming cells [SFC]) out of 1 × 10⁴ CTLs after expansion are shown. *P < .05 and **P < .01 by 2-tailed Student t test. (F) Results of ⁵¹Cr release assay showing antigen-specific, HLA-restricted cytotoxicity by amplified CTLs derived from WT1-TCR-transduced NSG-HLA-A24/HHD–recipient BM (A24-TCR-2) at indicated effector-to-target cell (E:T) ratios (upper). ⁵¹Cr release assays at an E:T ratio of 5:1 in the presence or absence of anti-HLA class I mAb or anti-HLA class II mAb (lower). (G) Cytotoxic activity of WT1-specific CTLs from A24-TCR-2 BM against leukemia cell lines (left). The cytotoxicity of these CTLs was inhibited by adding anti-HLA class I mAb, but not by adding anti-HLA class II mAb (right).