Hyperacute Rejection by Anti-Gal IgG1, IgG2a, and IgG2b Is Dependent on Complement and Fc-γ Receptors

Abstract

We have previously reported that anti-Gal-α1,3Gal (Gal) IgG3 mAbs mediate a classical complement-dependent hyperacute rejection (HAR), while anti-Gal IgG1 mAbs mediate HAR that is dependent on complement, the Fc-γ receptors FcγRII/III (CD32/CD16), and NK cells. IgG2a and IgG2b subclasses can activate complement and have FcγR binding properties in vitro. Whether these IgG subclasses can mediate HAR in vivo and the mechanisms by which they would do so are not known. In this study, we isolated spontaneous IgG switch mutants from an anti-Gal IgG1 hybridoma. In vitro complement-mediated hemolytic assays with mouse complement indicate that both anti-Gal IgG2a and IgG2b mAbs were more potent compared with the parent anti-Gal IgG1. In vivo administration of anti-Gal IgG2a and IgG2b mAbs into Gal−/− mice induced HAR of rat cardiac xenografts. HAR induced by anti-Gal IgG2a and IgG2b was dependent on complement activation and the presence of NK cells. Using FcγRIII-deficient (Gal−/−CD16−/−) recipients, we observed that HAR mediated by different anti-Gal IgG subclasses was variably dependent on FcγRIII, with IgG1 > IgG2b ≫ IgG2a = IgG3. Using FcγRI-deficient (Gal−/−CD64−/−) recipients, we observed that HAR mediated by anti-Gal IgG1, IgG2a, and IgG2b, but not by anti-Gal IgG3, was dependent on FcγRI. Collectively, these studies demonstrate the necessity and sufficiency of complement in IgG3-mediated HAR and the necessity of both complement and FcγR, especially FcγRI, in IgG1-, IgG2a-, and IgG2b-mediated HAR.

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