Complement C5 activation induces oxidation of cell surface PDI. (A) Binding of ATG to immobilized recombinant human PDI in comparison with rabbit IgG and polyclonal rabbit anti-PDI (αPDI). (B) Rabbit IgG and ATG were preincubated with recombinant human PDI in a 1:3 molar ratio and subsequently loaded on THP1 cells for 15 minutes at RT (100 µg/mL final concentration). Following washes, cellular binding of IgG and ATG was analyzed by indirect flow cytometry. A representative experiment is shown. (C) (Left) Rabbit IgG and ATG were preincubated with recombinant human PDI in a 1:3 molar ratio before PCA of IgG- and ATG-loaded THP1 cells was measured as described above (mean ± SD, n = 5). (Right) As a control experiment, recombinant PDI was added to THP1 cells after IgG or ATG loading (mean ± SD, n = 3). (D) IgG and ATG were preincubated with recombinant PDI as described above. Following loading on THP1 cells and exposure to NHP, annexin V-FITC binding was analyzed by flow cytometry. A (left) representative experiment and (right) summary statistics are shown (mean ± SD, n = 3). (E) (Left) IgG- or (right) ATG-loaded THP1 cells were exposed to NHP and subsequently analyzed for PDI antigen expression by single-color flow cytometry using monoclonal antibody 1D3 (αPDI) in comparison with an isotype-matched control. A representative experiment is shown. (F) ATG-loaded THP1 cells were exposed to NHP in the absence or presence of 50 µg/mL eculizumab (αC5) or blocking C7 monoclonal antibody (αC7). Following washes, cell-associated PDI activity was measured as described above (mean ± SD, n = 3).