Ectopic PKB activation augments IFN-α and TNF-α production. (A-B) CD34⁺ HPCs, retrovirally transduced with myrPKB or a control vector, were differentiated toward pDCs in 2 weeks. Cells were either left unstimulated, or HSV was added during the last 5 hours of culture. Expression of eGFP, CD123, BDCA-2, and IFN-α was analyzed by flow cytometry. The percentage of eGFP⁺CD123⁺BDCA-2⁺ pDCs producing IFN-α and the IFN-α staining intensity (MFI) on HSV-stimulated eGFP⁺CD123⁺BDCA-2⁺IFN-α⁺ pDCs was determined. Representative FACS plots showing eGFP⁺ pDC (A), and mean ± SEM percentage of IFN-α–producing pDCs and IFN-α staining intensity within the IFN-α⁺ pDCs (B) are shown (n = 3). (C) Peripheral blood pDCs were cultured in the presence or absence of VO-OHpic. After overnight preincubation, pDCs were cultured for 5 hours in medium, CpG-A, or Lox. Mean ± SEM percentage of IFN-α–producing pDCs of at least 4 independent experiments with different donors is shown. (D-E) CD34⁺ HPCs were cultured as for panels A and B. Cells were left without stimulus, or HSV or Lox was added during the last 5 hours of culture. The percentage of eGFP⁺CD123⁺BDCA-2⁺ pDCs producing TNF-α was determined. Representative FACS plots showing eGFP⁺ pDC (D) and mean ± SEM percentage of TNF-α–producing pDCs (E) are shown (n = 3). *P < .05, paired Student t test.