Hyperacute rejection by anti-Gal IgG1, IgG2a, and IgG2b is dependent on complement and Fc-γ receptors

We have previously reported that anti-Gal-alpha 1,3Gal (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-gamma, receptors Fc gamma RII/III (CD32/CD16), and NK cells. IgG2a and IgG2b subclasses can activate complement and have Fc gamma R 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 FcyRIII-deficient (Gal(-/-)CD16(-/-)) recipients, we observed that HAR mediated by different anti-Gal IgG subclasses was variably dependent on Fc gamma RIII, with IgG1 > IgG2b >> IgG2a = IgG3. Using Fc gamma RI-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 Fc,gamma RI. Collectively, these studies demonstrate the necessity and sufficiency of complement in IgG3-mediated HAR and the necessity of both complement and Fc gamma R, especially Fc gamma RI, in IgG1-, IgG2a-, and IgG2b-mediated HAR.