AML-associated Tregs reduced proliferation and IFN-γ secretion of adoptive transferred CTLs. (A) B6 mice were injected with 10⁶ C1498FFDsR cells. Congenic B6-ly5.2 (CD45.1⁺) CTLs (30 × 10⁶) were infused through an intravenous route 14 days after tumor injection or to naive mice. BrdU was added to the drinking water to track proliferation. Eighteen days after tumor injection, 4 mice per group were killed. Flow Cytometric Analysis (FACs) was done with liver leukocytes and splenocytes. Tumor-bearing mice had significantly reduced proliferation of CTLs compared with naive mice in the livers and spleens (P < .05). (B-C) CTLs (10⁶) and Tregs (10⁶) isolated from AML-bearing or naive mice were adoptively transferred to AML-bearing Rag^−/− mice. Thirteen days after transfer, FACs was performed on splenocytes (B) and liver leukocytes (C). Cells were gated on CD8 expression for CTLs. Intracellular IFN-γ expression was measured on gated CTLs. Tregs from AML-bearing mice significantly reduced the percentage of IFN-γ–producing CTLs in the spleen and liver. (D) Tregs isolated from AML-bearing or naive mice were cocultured with OT I CD8⁺ T cells stimulated with SIINFEKL peptide (Ovap) for 6 days. Cell supernatant was harvested and the IFN-γ level was determined. Tregs from AML-bearing mice inhibited IFN-γ production by OT I CD8⁺ T cells (P < .01), whereas naive Tregs were not suppressive. Results from 1 of 2 representative experiments are shown. Error bars represent SD.