PI3K-PKB-mTOR signaling is required for pDC development and survival. (A) CD34⁺ HPCs were differentiated toward pDCs in the presence of LY, VIII, Rapa, or their solvent dimethyl sulfoxide (DMSO). After 2 weeks, pDCs were identified by the expression of CD123, BDCA-2, and BDCA-4. Representative FACS plots and mean ± SEM percentage of pDCs standardized to control are shown. Data are representative of at least 3 independent experiments with different donors. (B) Peripheral blood pDCs were cultured with or without CpG-A in the presence or absence of LY, VIII, Rapa, or DMSO for 18 hours. S6 phosphorylation (p-S6) was determined. Representative FACS plots and mean ± SEM percentage of p-S6⁺ cells are shown (n = 4). (C-D) Peripheral blood pDCs were cultured with or without CpG-A in the presence or absence of LY, VIII, Rapa, or DMSO. Apoptosis was determined after 18 hours as the percentage of cells staining for annexin V and/or propidium iodide (PI). Apoptosis was standardized to control by subtracting the percentage of apoptotic cells in control cultures from the percentage of apoptotic cells in inhibitor cultures. Shown are representative FACS plots of unstimulated cultures (C) and mean ± SEM apoptosis standardized to control from at least 3 independent experiments with different donors (D). (E) LY, VIII, or Rapa was added to peripheral blood pDC cultures in increasing concentrations. DMSO concentration was similar in all cultures. Apoptosis was determined by Annexin V/PI staining after 18 hours and standardized to control as in panel D. Mean ± SD apoptosis in duplicate cultures is shown. (F) Peripheral blood pDCs were cultured with CpG-A in the presence or absence of LY, VIII, or Rapa (n = 3). Supernatants were harvested after 18 hours, and IFN-α concentrations were determined by ELISA and standardized to control cultures. Mean ± SEM concentrations are shown. *P < .05, paired Student t test.