CARTs derived from leukemia-bearing donors remain dysfunctional following in vitro activation, expansion and transduction. (A) Splenocytes from mice were enriched for CD3⁺ cells, which were used to generate mCD19 CARTs as described in methods. Representative dot plots showing PD1, Tim3, and LAG3 expression on after 4 days in culture. (B-C) Geometric mean of PD1 expression (B) and TIM3 expression (C) on mCD19 CAR-expressing T cells generated from healthy and leukemia-bearing donors. P < .05 at day 4 (Mann-Whitney U test). (D) C57BL/6 mice received 500 cGy radiation followed by tumor challenge with 10⁵ TCF3/PBX1.3. CARTs or mock (GFP+) T cells were generated from healthy and leukemia-bearing donors. CARs were administered IV at a dose of 3 × 10⁵ CARTs 3 days after tumor challenge, 500 cGy irradiation on day 3. Mock CAR provided no survival benefit compared with untreated mice, but mCD19 CAR resulted in 100% survival (P = .02, Mantel-Cox test). (E) Microarray was performed on mCD19 CARTs generated as in panel A on day 5 of culture. Heat map of representative gene expression associated with TCR signaling and T cell exhaustion is shown.