hOSCAR is expressed in myeloid cells. (A) Northern blot analysis. A hOSCAR-specific single band at approximately 1.8 kb was detected in peripheral blood leukocyte (PBL) and bone marrow. (B) RT-PCR analysis. Macrophages (MΦ) and granulocytes were derived from cord blood CD34⁺ progenitors. DCs were generated from cord blood CD34⁺ progenitors (CD34⁺ DCs) or from blood monocytes (mono-DCs). PBMCs, monocytes, blood T cells, and tonsillar B cells were freshly isolated. Activations were by CD40-ligand transfected L cells (for in vitro–derived DCs and B cells), LPS (for monocytes), PMA/ionomycin (for granulocytes, PBMCs, and MΦ), or anti-CD3, anti-CD28 antibodies (for T cells). hOSCAR and β-actin were amplified for 30 and 25 cycles, respectively. (C) Two-color flow cytometry analysis of PBMCs. Cells were labeled with biotin-conjugated anti-hOSCAR, followed by incubation with streptavidin-PE and FITC-conjugated mAbs against the indicated cellular markers. Acquisition was performed by gating on the different cell populations, depending on the forward and right-angle scatter parameters. (D) Four-color flow cytometry analysis of PBMCs. Cells were stained with FITC-lineage cocktail, PE-Cy5–anti-CD4, APC–anti-CD11c, and biotin-conjugated anti-hOSCAR, followed by streptavidin-PE. Primary DCs are included within lineage^–/CD4⁺ cells; CD11c⁺ cells correspond to myeloid DCs, whereas CD11c^– cells correspond to plasmacytoid DCs. (E) Cell surface expression of hOSCAR in monocytes and mono-DCs. Untreated monocytes (shaded histogram) and mono-DCs treated with LPS for 24 hours (solid bold) or untreated (shaded histogram) were analyzed by FACS for cell surface expression of hOSCAR, using anti-hOSCAR mAbs and GAM-PE. Dashed profiles indicate background staining with a control IgG1 mAb.