Anti–PD-L1 mAb treatment enhanced the resistance of adoptively transferred anti-AML CTLs to Treg-induced suppression, resulting in increased long-term survival. B6 mice (10 mice/group) were injected with 10⁶ C1498FFDsR cells followed by anti–PD-L1 mAb (every other day from days 10-20) and CTL treatment (day 14) as described. (A) Anti–PD-L1 mAb treatment alone significantly prolonged the survival of mice compared with either control mice or mice treated with CTLs alone, although all mice died of AML (▵ vs ■ or ▿, P < .01). Combined anti–PD-L1 mAb and anti-AML CTLs had a significantly greater survival benefit (P < .005) compared with either control mice (■) or mice receiving anti–PD-L1 mAb (▵) or CTL alone (▿). Combined therapy permitted 30% of mice with advanced AML to survive long-term. (B-C) B6 mice were injected with 10⁶ C1498FFDsR cells. A total of 30 × 10⁶ congenic B6-ly5.2 (CD45.1⁺) CTLs and anti–PD-L1 mAb treatment were given as described. BrdU was added to the drinking water to track proliferation. On days 20 or 25 after tumor injection, 4 mice per group were killed. Flow cytometry was done with liver leukocytes (B-C). (B) Anti–PD-L1 mAb treatment significantly augmented the percentage of BrdU⁺ adoptively transferred CTLs in the liver of mice compared with control mice. (C) Intracellular IFN-γ was determined for adoptively transferred CTLs in the liver of mice with advanced AML. Anti–PD-L1 mAb treatment significantly increased the percentage of IFN-γ–secreting CD8⁺ T cells. (D) Anti–PD-L1 mAb treatment did not alter the percentage of Foxp3⁺ Tregs found in the liver of AML-bearing mice. (E) A total of 10⁶ CTLs and 10⁶ Tregs isolated from AML-bearing mice were adoptively transferred to AML-bearing Rag^−/− mice. Anti–PD-L1 mAb was given as described. Thirteen days after CTL transfer, flow cytometric analysis was performed on adoptively transferred CTLs in the liver. Intracellular IFN-γ expression was measured on gated, transferred CTLs. Tregs obtained from AML-bearing primary recipients significantly reduced the percentage of IFN-γ, producing adoptively transferred CTLs in AML-bearing secondary recipients. Anti–PD-L1 mAb treatment significantly increased the percentage of IFN-γ–secreting transferred CTLs, similar to a level of transferred CTLs without Treg cotransfer. Results from one of 3 representative experiments are shown. Bar graphs represent mean ± SD.