FIG. 1.

Bone marrow (BM)-derived plasmacytoid dendritic cells (pDC) from pearl mice are defective for interferon-α (IFN-α) production in response to virus infections. CD34+ BM-derived cells from pearl and C57B6/L wild-type (wt) mice were purified by positive immunoselection and treated to generate conventional DC (cDC) or pDC, as previously described (Del Prete and others 2004). pDC were infected at multiplicity of infection (MOI) of 1 with murine cytomegalovirus (MCMV), Herpes Simplex virus (HSV)-2, Vesicular Stomatitis virus (VSV) or treated with CpG ODN 2216 (CpG-A) (20 μg/mL). [(A), left panel] After 24 h, total RNA was extracted, retrotranscribed with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) and real-time polymerase chain reaction performed with iQ^™ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) was carried out using primers specific for IFN-α (sense, 5′-CCACAGGATCACTGTGTACCTGAGA-3′; antisense, 5′-CTGATCACCTCCC AGGCACAG-3′). The cDNA was amplified for 45 cycles (95°C for 10 s and 60°C for 30 s). Results were normalized to the 18S signals and are presented as fold increase relative to the mRNA levels in unstimulated cells (assumed as the 1.0 value) (1 representative experiment out of 3). [(A), center and right panels] IFN-α and tumor necrosis factor-α (TNF-α) concentrations were measured by VeriKine Mouse IFN-α ELISA (PBL Interferon Source) and Duoset ELISA (R&D System) in the supernatants from BM-derived pDC stimulated as described above. (B) IFN-α and TNF-α concentrations were measured in the supernatants from BM-derived cDC infected with MCMV (MOI 1) or treated with lipopolysaccharide (LPS) (1 μg/mL) for 24 h. Data are expressed as mean±standard error of the mean (SEM) (n=3). Results were analyzed by the 2-tailed Student's t-test. *P<0.05.