Progressive leukemia induces phenotypic changes consistent with T-cell dysfunction. (A) Bone marrow from healthy C57BL/6 mice was analyzed by flow cytometry 12 days after challenge with 1 × 10⁵ viable or irradiated TCF3/PBX1.3. (B) Percentage of bone marrow CD4⁺ and CD8⁺ T cells expressing PD1 at the time points indicated. Line represents the mean, and error bars represent standard error of the mean. (C) Representative dot plots of bone marrow cells at day 12 following injection of TCF3/PBX1.3 compared with non–leukemia-bearing mice. Bottom panels represent gating on PD1⁺ CD4⁺ and CD8⁺ T cells. (D) Scatter plots showing percentage of PD1⁺ CD4⁺ and PD1⁺CD8⁺ bone marrow T cells expressing Tim3 and LAG3 at day 12 following injection of TCF3/PBX1.3 compared with non–leukemia-bearing mice. (P < .001, unpaired Student t test). (E) Leukemia-bearing mice were given 5 × 10⁶ T cells from mice bearing leukemia or vaccinated with irradiated TCF3/PBX1.3 (as in panel A). Mice received 10⁵ TCF3/PBX1.3 on day 0, 500 cGy irradiation on day 2 and adoptive transfer of T cells on day 2. Mice receiving T cells from vaccinated donors demonstrated survival benefit compared with leukemia-bearing donors (P < .0001, Gehan-Breslow-Wilcoxon test). (F) Percentage of CD8⁺ T cells expressing PD1, Tim3, and LAG3 in mice bearing MLL-AF4 ALL at day 23 following leukemia injection compared with non–leukemia-bearing irradiated mice. Control mice received 250 cGy irradiation. Line represents the mean, and error bars represent standard error of the mean.