Immunization of HLA class I Tg NSG recipients using a WT1 antigen. (A) Representative contour plots show development of WT1-specific CD8⁺ CTLs in PB from an HLA class I Tg NSG recipient before (left) and after (right) immunization using HLA-A*2402 WT1(mutant)_235-243 tetramers. (B) Frequencies of WT1-specific tetramer⁺ CTLs in the PB of HLA class I Tg NSG recipients were increased after immunization (n = 7). (C) Representative flow cytometry plots of tetramer assay in spleens from HLA class I Tg NSG recipients at day of euthanization. Data for the control group and the DC vaccine group in NSG-HLA-A2/HHD recipients (left 2 plots) and NSG-HLA-A24/HHD recipients (right 2 plots) are shown. (D) Summary of tetramer analysis in each organ at day of euthanization. Open inverted triangles, diamonds, and circles indicate the groups of no immunization control, immunized without DCs, and immunized with DCs, respectively. The frequencies of WT1 tetramer⁺ cells gated on CD8⁺ T cells in spleen, PB, BM, and MLN are plotted. (E) Representative contour plots of tetramer assay before and after in vitro expansion of CTLs derived from spleens of HLA class I Tg NSG recipients in the group immunized with DCs. Isotype control (upper) and WT1 tetramer (lower). (F) Frequencies of WT1 tetramer⁺ spleen-derived CTLs (%) before and after in vitro expansion. (G) Fold increase of tetramer⁺ CTL numbers before and after in vitro expansion. Bars indicate the mean value. *P < .05 and **P < .01 by 2-tailed Student t test. Ctrl, control.