Normal circulating pDCs express CD33 at similar levels as granulocytes. (A) Identification of circulating pDCs using a 3-color staining. DCs were identified as lineage-negative (Lin neg.) CD4⁺ cells (gate R1). Lymphoid DCs express CD123^high, BDCA-2, but not CD11c (circles). (B) CD33 expression on normal pDCs. Because BDCA-2 staining allows differentiation of lymphoid DCs (positive cells) from other cells,^5,36  BDCA-2 mAb was used simultaneously with anti-CD33 mAb to determine CD33 expression on circulating pDCs. A better detection of CD33 was observed using PE-conjugated mAb (LeuM9 or WM53) than FITC mAb (WM-54 or D3HL60.251). Results are expressed as rfi or MFI R (MFI obtained with anti-CD33 mAb/MFI obtained with isotype control mAb). For the 6 donors analyzed, the number of events in the gate R2 varies from 170 to 200 (198 in the representative dot plot). (C) Comparison of CD33 level expression on peripheral blood cells. CD33 expression on pDCs was compared with the expression on granulocytes (CD33^low cells,^20,21  MFI R = 15), on monocytes (CD33^high cells,^20,21  MFI R = 182) and on lymphocytes (CD33 nonexpressing cells, MFI R = 1) from the same sample. Granulocytes, monocytes, and lymphocytes were identified by forward light scatter (FSC)/side light scatter (SSC) gating. Lymphoid DCs were identified as BDCA-2⁺ cells. Cells were stained with either isotype control (dotted line) or anti-CD33 mAb LeuM9 (gray hatched curve) and analyzed by flow cytometry.