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Anti-HLA antibodies with complementary and synergistic interaction
strong variation in the level of complement deposition of C3b and C4b was observed between donors, whose vari- ous HLA type matched in all cases with the specificity of WIM8E5 (Figure 1B and D). A gene dosage effect was observed; donors with two HLA-A antigen specificities compatible with WIM8E5 binding induced more C3b dep- osition compared to donors expressing one compatible HLA-A antigen and HLA-A2 (with reduced binding or
affinity) (Figure 1E). Only minor C3b deposition was observed when platelets from donors expressing only one compatible HLA-A antigen (e.g., A11 and A32) were used (Figure 1E right panel). Variable levels of C3b deposition on platelets of 5 donors expressing HLA-A2 in combina- tion with another WIM8E5 HLA-A-compatible antigen were observed that correlated with WIM8E5 binding (Figure 1F) This suggests that the level of IgG opsoniza-
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Figure 3. Complement deposition on platelets by HLA mAbs occurs via the classical complement pathway. (A) Pre-incubation of platelets with 50 mg/ml anti-C1q inhibits C3b deposition (n=7), (B) and C4b deposition (n=7). (C) Dose response of increasing concentrations anti-C1q to inhibit C3b deposition induced by 20 mg/ml WIM8E5 (n=4) (D) Effect of increasing concentrations of Fc:Fc blocking peptide DCAWHLGELVWCT on complement deposition induced by WIM8E5 (20 mg/ml) was measured by C3b deposition (n=3), (E) and C4b deposition (n=4). (F) Effect of 200 mg/ml Fc:Fc blocking peptide on C3b deposition by WIM8E5 and SN607D8/SN230G6 was compared to the irrelevant control peptide GWTVFQKRLDGSV (n=4). *P<0.05, **P<0.01.
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