SRA/CD204 silencing enhances immunostimulatory capability of TLR4-activated DCs. (A) Lentivirus-mediated RNA interference efficiently down-regulates SRA/CD204 expression in DCs. BM-DCs (105 cells/well) were transfected with LV-scramble shRNA, LV-SRA shRNA at a multiplicity of infection of 50, or left untreated. Cells were harvested 2 days later and analyzed by immunoblotting using anti-SRA/CD204 antibodies. (B) Silencing of SRA/CD204 in DCs by lentivirus encoding SRA shRNA, as indicated by FACs analysis. (C) SRA/CD204 silencing enhances ability of DCs to prime naive OT-I cells. BM-DC cells were transfected with LV-SRA shRNA, LV-scramble shRNA, or left untreated. DCs were harvested 2 days after infection and pulsed with OVA protein. After LPS stimulation and extensive washes, DCs were cocultured with OT-I cells at different ratios. OT-I cell proliferation was measured by 3H-thymidine incorporation assays. (D) Increased IL-2 production by OT-I cells stimulated with SRA/CD204-silenced DCs. After incubation, the levels of IL-2 in the supernatant were determined using an ELISA (*P < .005, SRA shRNA vs scramble control). (E) SRA-silenced DCs were stimulated with or without LPS, followed by coculture with OT-I cells at a ratio of 1:10. T-cell proliferation was assayed by 3H-thymidine uptake (*P < .005, SRA shRNA vs control). Representative results from 2 independent experiments are shown. Error bars indicate SE.
