ID3 ablation in human T cells reduces xeno-GVHD progression while preserving antitumor activity. ID3 was knocked out from human T cells (including both CD4⁺ T and CD8⁺ T cells) using CRISPR-Cas9 technology and maintained in culture for 7 to 9 days. NSG mice were IV infused with T-cell depleted peripheral blood mononuclear cells (TCD-PBMCs; 10 × 10⁶ cells per mouse) alone or together with WT or ID3-CRISPR-KO human CD4⁺ T cells (10 × 10⁶ cells per mouse) and CD8⁺ T cells (10 × 10⁶ cells per mouse). (A) Survival rates of NSG mice receiving WT and ID3-CRISPR-KO human T cells. (B) The overall survival of leukemia-bearing mice treated with or without WT or ID3-CRISPR-KO CD19-CAR T cells (n = 8-12). (C-F) Balb/c mice were subjected to total body irradiation (4.5 Gy on day −1 and 4 Gy on day 0) followed by infusion of 5 × 10⁶ B6 TCD-BM alone or together with 3 × 10⁵ B6 (CD45.1^-CD45.2⁺) naïve WT or Id3-cKO CD4⁺ T cells and 4 × 10⁵ WT or Id3-cKO CD8⁺ T cells. Recipient mice were challenged with luciferase-expressing P815 mastocytoma cells or A20 lymphoma cells before T-cell infusion. (C) Survival probability. (D) Representative images of tumor luciferase activity detected with live animal in vivo imaging system (IVIS) on days 7, 14, and 34 after transplantation. (E) Summary of GVHD death and tumor death in allo-HSCT mice challenged by P815 cells and A20 cells. (C-E) All Balb/c mice receiving TCD-BM died from tumor; WT donor T cells induced antitumor activity with prolonged median survival time (27.5 days), but all succumbed to tumor and/or GVHD by day 38; in contrast, donor T cells lacking Id3 preserved beneficial GVT effects, leading to significantly improved survival of tumor mice undergoing allo-HSCT. (F) Survival of animals challenged with A20 lymphoma cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. GVT, graft-versus-tumor.