CAL-1 cells closely resemble primary pDCs. (A) The leukemic pDC cell line CAL-1 expresses transcripts commonly present in freshly isolated primary pDCs, including Spi-B, E2-2, IRF-7, TLR-7, TLR-9, and BDCA2 as shown by semiquantitative RT-PCR. The housekeeping genes GAPDH and actin are shown as loading controls. (B) Flow cytometric analysis of CAL-1 cells after staining with antibodies directed against the cell surface markers BDCA2, CD123, CCR7, CD45RA, CD62L, and CD56 (black lines). Isotype control stainings are shown as gray filled histograms. (C) Surface expression of the costimulatory molecules CD40, CD80, CD86, and HLA-DR was measured by flow cytometry on unstimulated CAL-1 (gray lines) and after 20 hours stimulation with CpG-B (black lines; isotype control stainings are shown as gray filled histograms). (D) CAL-1 cells were stimulated for 4 hours with CpG-B or left unstimulated in medium only, and gene expression levels of CD40, IFN-α and IFN-β1 were measured in stimulated versus unstimulated cells by qPCR. (E) CAL-1 cells were cultured in the presence of CpG-B or medium for 6 hours. Culture supernatants were analyzed for the presence of TNF-α and IL-6 by cytokine bead array analysis. ND indicates not detectable (below detection sensitivity of the assay of 4 pg/mL).