Figure 8.

C5aR1 deficiency impaired pDCs function and differentiation in vitro. (A,B) pDCs were isolated from splenocytes of C5aR1^+/+ and C5aR1^−/− mice. Culture medium was used as a control. (A) Purified pDCs (1 × 10⁶/ml) were stimulated with IMQ or R848 (10 μg/mL) in vitro for 24 hr. The levels of IFN-α and TNF-α in the culture supernatants were measured by ELISA. (B) pDCs (1 × 10⁶/ml) in transwell plates were exposed to C5a (42 nM) for 90 min. Cells that migrated to the lower chamber were measured. The purity of isolated pDCs and IMQ-induced C5aR1 expression of isolated pDCs are shown in Figure S9. C5a levels in cultural supernatant of purified pDCs and in serum of naïve C5aR1^+/+ or C5aR1^−/− mice are shown in Figure S10. (C) BM cells from C5aR1^+/+ and C5aR1^−/− mice were stimulated with recombinant mouse FLT3L (200 ng/ml for 10 days and the frequency of pDCs was measured by FCM. Left, a representative FCM graph. Right, average percentages and MFIs of PDCA-1⁺ cells. (D) BM cells from C5aR1^+/+ mice were stimulated with recombinant mouse FLT3L (200 ng/mL) alone or with C5a (42 nM) for 10 days, culture medium was used as a control. The frequency of pDCs was analyzed by FCM. Values are presented as mean ± SEM and data were obtained from at least three independent experiments. ^**p < 0.01; ^***p < 0.001.