Transplant of WT1-TCR gene–transduced HSCs into NSG-HLA-A24/HHD recipients. (A) Purification of CB CD34⁺CD38⁻ cells using a CD34⁺ cell–enriched CB sample (left). WT1-TCR Vα and Vβ genes were transduced into the isolated cells with a lentiviral vector at a multiplicity of infection of 100. After 5 days of incubation, GFP⁺ cells were sorted and transplanted into NSG-HLA-A24/HHD newborn mice (right). (B) Multilineage analysis of WT1-TCR gene–transduced NSG-HLA-A24/HHD recipients. Representative plots of spleen (upper) and thymus (lower) are shown. (C) Frequencies of B cells and T cells gated on human CD45⁺ leukocytes in the spleen and thymus of NSG-HLA-A24/HHD recipients transplanted with nontransduced/GFP control–transduced or WT1-TCR gene–transduced HSCs. (D) Frequencies of human T-cell subsets in the spleen and thymus of NSG-HLA-A24/HHD WT1-TCR gene–transduced HSC recipients (spleen: [n = 6 nontransduced or GFP control recipients] CD4⁺, 64.3% ± 3.1% and CD8⁺, 28.5% ± 3.5%; [n = 11 WT1-TCR gene–transduced HSC recipients] CD4⁺, 50.8% ± 7.4% and CD8⁺, 29.0% ± 5.3%; thymus: [n = 5 nontransduced or GFP control recipients] CD4⁺, 46.7% ± 7.1%; CD8⁺, 29.7% ± 3.8%; and double-positive (DP), 20.4% ± 5.6%; [n = 15 WT1-TCR gene–transduced HSC recipients] CD4⁺, 25.1% ± 3.8%; CD8⁺, 35.2% ± 4.3%; and DP, 33.6% ± 6.6%). Open and closed circles indicate nontransduced and GFP control recipients, respectively. Bars indicate the mean value. FSC, forward scatter.