PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function by Lianne van de Laar, Aniek van den Bosch, André Boonstra, Rekha S. Binda, Miranda Buitenhuis, Harry L. A. Janssen, Paul J. Coffer, and Andrea M. Woltman Blood Volume 120(25):4982-4991 December 13, 2012 ©2012 by American Society of Hematology

PI3K-PKB-mTOR signaling is required for pDC development and survival. 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. Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology

Enhanced PI3K-PKB activity improves CD34-derived pDC development. Enhanced PI3K-PKB activity improves CD34-derived pDC development. (A) CD34+ HPCs were differentiated toward pDCs in the presence or absence of VO-OHpic. After 2 weeks, cells were counted with trypan blue exclusion and analyzed for the expression of CD123, BDCA-2, and BDCA-4 by flow cytometry. Absolute numbers of pDCs per well were calculated, and the fold increase of pDCs in inhibitor cultures compared with control cultures was determined. Representative FACS plots showing pDC percentages and mean ± SEM absolute pDC numbers are shown (n = 6). (B) CD34+ HPCs, retrovirally transduced with myrPKB or a control vector, were differentiated toward pDCs. The percentage of eGFP+ cells was analyzed 2 days after transduction to determine the transduction efficiency. After 2 weeks, cells were counted with trypan blue exclusion and analyzed for eGFP, CD123, BDCA-2, and BDCA-4 by flow cytometry. Absolute numbers of eGFP+ pDCs per well were determined and corrected for the difference in transduction efficiency. The fold increase of pDCs in myrPKB cultures compared with control cultures was determined. Representative FACS plots showing pDCs within the eGFP+ population and mean ± SEM absolute eGFP+ pDC numbers are shown (n = 6). *P < .05, Wilcoxon signed rank test. Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology

Ectopic PKB activation increases costimulatory molecule expression by pDCs. Ectopic PKB activation increases costimulatory molecule expression by pDCs. CD34+ HPCs, retrovirally transduced with myrPKB or a control vector, were differentiated toward pDCs in 2 weeks. Cells were either left unstimulated, or CpG-A or Lox was added during the last 18 hours of culture. Expression of HLA-DR, CD40, and CD86 by eGFP+CD123+BDCA-2+BDCA-4+ pDCs was determined. Specific staining was calculated by subtracting the mean fluorescence intensity (MFI) of the isotype control from the MFI measured for each marker (n = 3). (A) Representative FACS plots gated on eGFP+ pDCs are shown. Numbers in the FACS plots indicate the specific staining calculated for the experiment shown. (B) Mean ± SEM specific staining of eGFP+ pDC is shown. *P < .05, paired Student t test. Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology

Constitutively active PKB increases development and activation status of CD34-derived pDCs in vivo. Constitutively active PKB increases development and activation status of CD34-derived pDCs in vivo. CD34+ HPCs were retrovirally transduced with myrPKB or a control vector. Day 3 unsorted cells were intravenously injected into β2-microglobulin−/− NOD/SCID mice. Six weeks after transplantation, bone marrow cells were analyzed for the expression of eGFP, CD123, BDCA-2, and CD86. The presence of CD123+BDCA-2+ human pDCs within the eGFP+ fraction and the expression of CD86 by eGFP+CD123+BDCA-2+ human pDCs was determined. (A) Shown are representative FACS plots gated on eGFP+ cells and percentage CD123+BDCA-2+ human pDCs within the eGFP+ population per mouse (control, n = 4; myrPKB, n = 5). (B) Specific CD86 staining of eGFP+ human pDCs was calculated by subtracting MFI of the isotype control from the MFI measured for CD86. Expression in different experiments was standardized to control per experiment. Shown are representative FACS plots gated on eGFP+ human pDCs and standardized CD86 expression by eGFP+ human pDCs per mouse (control, n = 3; myrPKB, n = 3). Numbers in the FACS plots indicate the specific staining calculated for the experiment shown. *P < .05, Mann-Whitney U test. Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology

Ectopic PKB activation augments IFN-α and TNF-α production. 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. Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology

Enhanced IRF7 and p65 phosphorylation in myrPKB-expressing pDCs. Enhanced IRF7 and p65 phosphorylation in myrPKB-expressing pDCs. CD34+ HPCs were transduced and cultured as described in the legend for Figure 5. HSV was added during the last 5 hours of culture. Expression of eGFP, CD123, BDCA-2, phosphorylated IRF7 (p-IRF7), and phosphorylated p65 (p-p65) was analyzed by flow cytometry. The percentage of eGFP+CD123+BDCA-2+ pDCs expressing p-IRF7 (A) or p-p65 (B) was determined. Representative FACS plots showing eGFP+ pDCs and percentages per independent experiment are shown (n = 3). Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology

Decreased S6 phosphorylation in pDCs from HBepos chronic hepatitis B patients. Decreased S6 phosphorylation in pDCs from HBepos chronic hepatitis B patients. PBMCs from healthy subjects (HC), HBepos and HBeneg chronic hepatitis B patients (all matched on age and sex; HBeneg and HBepos patients matched for serum ALT levels and HBV viral load) were cultured for 5 hours. CpG-A or Lox was added 4 hours, 2 hours, 1 hour, or 15 minutes before the end of culture. S6 phosphorylation (p-S6) was analyzed within the CD11c−BDCA-4+ pDC population. Results are representative for 6 (15-minute, 2-hour, 4-hour stimulation) or 8 (medium and 1-hour stimulation) subjects per group. (A) Representative FACS plots and percentages p-S6+ pDCs of unstimulated and 1-hour stimulated pDCs are shown. (B) Mean ± SEM percentages p-S6+ pDCs are shown for each condition. Unstimulated cultures are represented by 0. (C) Percentages p-S6+ pDCs of unstimulated pDCs, and the proportion of unstimulated pDCs within the PBMC population were determined. Data from HC, HBeneg, and HBepos patients were combined, and the Pearson rank correlation coefficient (rp) was calculated. Significance and rp of the correlation are indicated. *P < .05, unpaired t test compared with HC. Lianne van de Laar et al. Blood 2012;120:4982-4991 ©2012 by American Society of Hematology