Figure 3

C'-ADE is mediated by CR2 and can also be mediated by a mutant CR2 lacking a cytoplasmic tail. (A) Binding of two anti-CR2 mAbs, 1048 and 1F8, to SupT1/R5 cells was tested by flow cytometry. Both antibodies recognise a C3d-binding region-containing portion of CR2, but only 1048 blocks ligand binding. Both are shown to bind SupT1/R5 cells, at 10 μg/ml. (B) Left panel: the involvement of CR2 in C'-ADE was initially investigated by pre-incubating SupT1/R5 cells with increasing concentrations of C3dg ligand-blocking mAb 1048, and non-blocking mAb 1F8 as a control, before performing enhancement assays as usual, with MM34.443 virus and day 443 autologous serum. Right panel: a further three subjects (MM24.464 virus with day 292 serum; MM27.585 virus with day 299 serum; MM32.10 virus with day 21 serum) were tested in the presence of 10 μg/ml 1F8 or 1048 mAb. All experiments were performed in the presence of C', and fold enhancement was calculated relative to infection in the presence of NHS + C' and in the absence of anti-CR2 mAbs. (C) NP2/CD4/R5 cells stably expressing CR2 and CR2ΔCT were established by retroviral vector transduction. Expression levels were determined by flow cytometry using an anti-CR2 mAb. (D) Enhancement experiments were carried out on NP2/CD4/R5 (control; ctrl), NP2/CD4/R5/CR2 (CR2) and NP2/CD4/R5/CR2Δcytoplasmic tail (ΔCT) cells using MM24.464 virus opsonised with day 292 autologous serum, MM32.10 virus opsonised with day 15 autologous serum, and MM33.12 virus opsonised with day 719 autologous serum, and HIC' (white bars) or C' (black bars). Fold enhancement was calculated relative to infection in the presence of NHS + HIC' or NHS + C', as appropriate, on each cell line. Error bars represent standard deviations from 3 experiments. (E) Images of the enhanced infection of MM33.12 virus by day 719 autologous serum on NP2/CD4/R5/CR2 cells. The left-hand image shows infection in the presence of NHS + C', and the right-hand image infection in the presence of day 719 serum + C'.