HLA antibody epitope and clonality are important determinants of capacity to fix complement in in vitro clinical and functional assays

We tested human allele-specific HLA class I antibodies (Ab) alone and in combination for their capacity to activate complement (C’) in in vitro assays. Methods Human HLA I Ab (clones SN607D8, SN230G6, MUL2C6, all IgG1) were diluted (0.01–0.5  μ g/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 cells was measured by flow cytometry, and cytotoxicity was measure using standard rabbit (rb-CDC) and human C′ (hum-CDC) assays. Results At 500 ng/mL, a pan HLA I Ab recognized all 97 beads with a mean IgG-MFI of around 5000, while anti-A2/B17 IgG1 at the same concentration bound only 6 beads (mean IgG-MFI u003e 20000 on those beads), and anti-A3/A11 bound 8 beads (mean IgG-MFI 17000). PRA beads displayed a similar phenomenon, pointing to a dilution effect of Ab with broad specificities. Anti-A2/B17 IgG1 bound to A2, B57 and B58 beads with uniform IgG-MFI and C1q-MFI u003e 1000. Anti-A3/A11 bound A1, A3, A11, A24, A36 and A80, with highest IgG-MFI on A11 and lowest on A36 (approximately 25% lower than A11). C1q-MFI was positive for all beads except A36, even at the highest concentration tested, and this bead had the lowest antigen density. Dilution of anti-A3/A11 IgG1 led to negative C1q results on A1 and A3, but A11 and A24 remained positive, suggesting that these antigens are crossreactive to the high affinity antigens A11 and A24. Anti-A2/A28 bound to all A2, A68 and A69 beads, with lower IgG-MFI on A∗02:03. C1q was positive ( u003e 1000 MFI) on all but A∗02:03, which differs from the other alleles at 149 AAH and 151HV epitopes. C1q-MFI and C3d-MFI on the A2 beads (including A∗02:03) was dramatically increased when two clones recognizing HLA-A2 (anti-A2/A28 mixed with anti-A2/B17) were present compared with each clone alone at the same total IgG concentration. Dramatically more C4d was deposited on A2+ cells by 2 anti-A2 clones vs. each alone. While anti-A3/A11 IgG1 caused lysis of A3+ B cells in rb-CDC, no cytotoxicity was observed with human C′. Conclusions Our results illustrate several factors (Ab affinity, reactivity, and antigen density) influencing C1q fixation in C1qSreen. Additionally, these results highlight the synergistic effect of Ab in close proximity on C’ activation and may explain some instances of low IgG-MFI but strong C′ activation.