Figure 1

CD40 and CD80 gene silencing in vitro . (A & B) In vitro gene silencing determined by quantitative RT-PCR. C57BL/6 mice bone marrow DCs were cultured for 6 days and were transfected with CD40, CD80 or scrambled siRNA using lipofectamine 2000. Non-transfected cells served as a negative control. Twenty-four hours after transfection, LPS was added for another 24h. Forty-eight hours after transfection, cells were harvested and total RNA was extracted. Transcripts of CD40 (A) and CD80 (B) were determined using quantitative RT-PCR. (*p < 0.01, CD40 or CD80 siRNA vs untransfected or scrambled siRNA transfected cells). (C & D) In vitro gene silencing of CD40 and CD80 detected by flow cytometry DCs were culture and transfected with siRNA as described in A &B. DCs were harvested and stained with FITC-labeled CD40 and PE-labeled CD80 antibodies. The expression of CD40 (C) and CD80 (D) was detected by flow cytometry. (E) CD40 and CD80 silenced DCs attenuate allogeneic T cell proliferation. Bone marrow DCs were cultured and transfected with CD40 and CD80 siRNA as described in A &B. Forty-eight hours after transfection, DCs were collected and co-cultured with allogeneic T cells in a 96 well plate at various ratios as indicated. [³H] was added 48h after co-culture, and its incorporation was measured as an indicator of T cell proliferation. (*p < 0.01 vs control group).