Evaluation of the pDCs and mDCs in WHIM patients. (A) Percentages of mDCs and pDCs (calculated as the percentage of the lymphocyte population) in 5 WHIM patients compared with 24 healthy subjects. To identify pDCs and mDCs via flow cytometry, we used the following gating strategy. Side scatter and staining with anti-CD4-peridinin chlorophyll protein and anti-CD14/anti-CD15/anti-CD19/anti-CD20-fluorescein isothiocyanate were used to exclude B cells, neutrophils, and monocytes. (B) IFN-α production was evaluated after HSV1 infection (dose ranging from 10 to 10 000 pfu/mL, for 24 hours; B) or CpG (Toll-like receptor 9 ligand, 5μM for 24 hours) stimulation (C) in PBMCs of 3 WHIM patients compared with healthy subjects. PBMCs (2.5 × 10⁶/mL) were plated in U-bottomed 96-well plates and cultured with 5μM CpG (ODN2216, InvivoGen) or infected with HSV1 (strain MacIntyre; from 10 up to 10 000 pfu/mL) for 24 hours. IFN-α levels in culture supernatants were determined using a VeriKine human IFN-α ELISA Kit (PBL InterferonSource) according to the manufacturer's instructions. (B-C) Results are presented as average of duplicate data. Error bars represent SE. Comparison of cytokine production by nonparametric analysis shows significant differences between WHIM patients and control subjects.