PD1 expression induced by progressive ALL is partially TCR independent and does not define functional impairment, nor does blockade of inhibitory receptors prevent or reverse dysfunction. (A) CD8⁺ T cells from the bone marrow and spleens of OT1/RAG^−/− TCR transgenic mice were analyzed by flow cytometry for PD1 expression 10 days after injection of 1 × 10⁵ TCF3/PBX1.3 challenge. (P < .01, unpaired Student t test). (B) Representative zebra plots of mice represented in panel A. (C) CD8⁺ T cells from the bone marrow and spleens of OT1/RAG^−/− TCR transgenic mice harvested and enriched using a T-cell selection column 14 days after injection with TCF3/E2aPBX1.3 and transferred into secondary recipients (1 × 10⁶/recipient) 2 days after injection with ovalbumin-transduced TCF3/E2aPBX1.3. Fourteen days later, bone marrow analyzed by flow cytometry for ovalbumin-expressing CD19⁺ B220⁺ cells. (D) Experimental design as in panel D with the exception that OT1 donors were irradiated (250 cGy) and injected with MLL-AF4 and T cells were harvested 21 days later. Non–leukemia-bearing donors also received irradiation. (E) Leukemia-bearing mice were given a subcurative dose (1 × 10⁶) of sorted PD1⁺ or PD1⁻ T cells from mice bearing leukemia or vaccinated with irradiated TCF3/PBX1.3. RAG1 mice received 10⁵ TCF3/PBX1.3 on day 0 and adoptive transfer of T cells on day 2. (F) Anti-PD1 (200 µg/dose) and/or anti-PDL1 (200 µg /dose) was administered intraperitoneally every 3 days beginning 1 day after 10⁵ TCF3/PBX1.3 challenge. (G) Anti-TIM3 (250 μg/dose) and/or anti-PD1 was administered as in panel D. (H) Splenic CD8⁺ T cells were collected from irradiated TCF3/PBX1.3 vaccinated mice (as in Figure 3G) and administered with or without PD1 and/or TIM3 blockade. Antibody administration was initiated 1 day prior to T-cell transfer and continued for up to 5 weeks (P < .0001, Mantel-Cox test).