OR5 Igg subclass and concentration are determinants of HLA class I antibody capacity to fix complement in in vitro clinical and functional assays

Abstract

It has been presupposed that “complement (C’) fixing” HLA antibodies (Ab) detected by C1qScreen or CDC are IgG1 or IgG3. We tested monoclonal HLA Ab of each human IgG subclass to evaluate the effect of subclass in in vitro complement assays. Chimeric HLA I Ab carrying the same variable region (pan HLA I) and human IgG1–4 constant regions were diluted (0.01–20 μg/mL) in human serum containing no HLA Ab, and tested in One Lambda LabScreen, C1qScreen, and Immucor LifeCodes Single Antigen and C3d Assays. C4d deposition on B cells and human endothelium was measured by flow cytometry, and cytotoxicity was measure using standard rabbit (rb-CDC) and human C’ (hum-CDC) assays. IgG-MFI, C1q-MFI, and C3d-MFI were dependent upon Ab concentration; however, the dynamic linear ranges of these assays was quite different. IgG-MFI reached saturation at lower Ab concentrations (500 ng/mL for IgG1) than C1q-MFI, while C1q-MFI became negative at Ab concentrations (0.125 ng/mL) still detectable by IgG-MFI. Lower amounts of Ab, including IgG2, were detectable by C3d assay than by C1qScreen. Ab concentration correlated well with C1q-MFI and C3d positivity. There was no linear relationship between IgG-MFI and C1q-MFI for any subclass, but C3d deposition did correlate with IgG-MFI. A threshold for C1q positivity was observed which differed for each subclass; for example, 15000 IgG1-MFI translated to C1q-MFI > 1000. Beads with lowest antigen density often had lowest IgG-MFI, C1q-MFI and C3d-MFI signals, with some false negative C’ results. Subclass-specific differences in C’ activation were observed. IgG2 triggered unanticipated strong C1q deposition in C1qScreen, and was nearly as potent as IgG1, but had lower potency in the C3d assay. Positive rb-CDC reactions were observed with IgG1, IgG3, and, unexpectedly, IgG2. In the hum-CDC, no chimeric subclass caused a positive reaction. Deposition of human C4d on the surface of cells could be detected, in a dose- and subclass-dependent manner. Our results highlight the dependence of C’ fixation and activation on Ab subclass and concentration, and illuminate important caveats to interpreting these assays. IgG-MFI did not directly correlate with C1q-MFI, there was a linear relationship between Ab concentration and C’ deposition in both C1qScreen and the C3d assay.