ATP-competitive dual mTORC1 and 2 inhibition dose-dependently and selectively upregulates DC B7-H1 expression. B6 mouse BM-derived conventional DCs were differentiated in the presence of vehicle (DMSO) or the indicated mTOR inhibitor (10 ng/mL RAPA or various concentrations of Torin1 or AZD8055). (A) Forward scatter (FSC) vs side scatter (SSC) flow cytometry plots of CD11c⁺-purified DCs. (B) CD11c-gated cells were analyzed for CD86, B7-H1 (PD-1 ligand 1), and B7-DC (PD-1 ligand 2) expression by flow cytometry in unstimulated cultures and cultures stimulated with LPS on day 7 for 18 hours. Isotype controls are indicated by the shaded histogram; unstimulated (gray line) and LPS-stimulated cells (black line) are also shown. The percent of cells staining positive and the MFI are indicated in the upper left and right corners, respectively. (C) The frequency of CD11c⁺ DCs in BM cell cultures was determined on day 8. (D) mTOR inhibition reduced the yield of CD11c⁺ DCs isolated from BM cell cultures on day 8. Viable cell numbers were determined by trypan blue exclusion. (E) Quantification of CD86, B7-H1, and B7-DC expression (MFI) across multiple experiments. (F) CD11c-gated cells from BM cultures exposed to increasing concentrations of AZD8055 (400, 800, and 1200 nM) were assessed for CD86 and B7-H1 expression. (G) B7-H1 expression on wild-type (WT) or rictor^−/− BM-derived DCs. Percent positive cells and MFI are indicated in the upper left and right corners, respectively. (H) Quantification of panel G across multiple experiments. Bar graph values are normalized to WT or the DMSO treatment condition. n ≥ 3 experiments for all data presented. *, # P < .05 when compared with control and RAPA, respectively.