Figure 5.
C3a promotes plasma-derived FVIII plus lipopolysaccharide-mediated T-cell responses. (A) Untreated dendritic cells (DC) and freshly isolated T cells were stained with antibodies directed against C3aR and C5aR (solid lines), respective isotype controls (dashed lines), or were left unstained (grey-shaded curves), and analyzed by fluorescence-activated cell sorting (FACS). Overlay histograms of 1 representative donor are shown. (B) DC were treated for 24 hours with plasma-derived (pd)FVIII (1 IU/mL) plus lipopolysaccharide (LPS) (0.1 mg/mL) in the presence or absence of the following complement inhibitors (inhib) or controls (ctrl): anti-C3/C3a/C3adesArg (αC3/C3a, n=16), anti-C5/C5a/C5adesArg (αC5/C5a, n=10), isotype control antibody (at 2 or 5 mg/mL), C3-targeting peptide inhibitor compstatin (n=7), control peptide (OVA323-339, both at 1 mg/mL), or anti-C5 (eculizumab, at 2 mg/mL, n=11). On day 9 of co-culture with PKH26-labeled autologous T cells, T-cell proliferation was assessed by FACS. Shown are the percentages of proliferated T cells from at least 3 independent experiments. (C) and (D) DC were pretreated for 1 hour with antagonists for C3aR (C3aRA) or C5aR (C5aRA) at the indicated concentrations followed by treatment with pdFVIII (1 IU/mL) plus LPS (0.1 mg/mL). Control DC were pretreated with dimethyl sulfoxide (DMSO) prior to stimulation with pdFVIII plus LPS. 24 hours later, PKH26-labeled auto-logous T cells were added and co-cultured for 9 days before the percentages of proliferated PKH26-labeled T cells were analyzed by flow cytometry. (C) FACS plots of 1 representative donor are given for C3aRA applied at 10 mM and C5aRA at 0.1 mM. (D) Summarized data of several donors (n=8) from 3 independent experiments are shown. Statistical significance was determined using the Wilcoxon matched-pairs signed rank test. Error bars indicate the means ± standard error of the mean.*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns: not significant. SSC: side scatter; FSC: forward scatter.