Anti-Gal IgG1 and IgG3 monoclonal antibodies have different abilities to induce xenograft rejection
There is increasing evidence that elicited xenoreactive antibodies (XAbs), especially IgG, play a major role in the acute xenograft rejection. We have produced a series of induced anti-Gal hybridomas from α1,3galactosyltransferase-knockout (GT-Ko) mice after rat to mouse heterotopic cardiac xenografting. In this study, we have characterized the differential ability of two anti-Gal IgG monoclonal antibodies (mAbs), of the IgG1 and IgG3 subclass, to elicit xenograft rejection. Analysis of the VH encoding the anti-Gal IgG3 (GN4-31) indicated that it was derived from the VH606-14 A gene and it contained 7/1 R/S mutations. The IgG1 (GT6-27-23) clone was also derived from the VH606-14 A gene and it contained 13/6 R/S mutations. The affinity of the IgG3 clone (5 × 10−7 M) was seven-fold lower that the IgG1 (7 × 10−9 M). In vitro complement-meditated lysis assay confirmed that only anti-Gal IgG1, but not IgG3, could cause the lysis of pig red blood cells. Neither anti-Gal IgG could directly mediate antibody-dependent cell lysis with pig PK15 cells as targets, and GT-Ko splenocytes as effector cells. We have also developed a new model of xenotransplantation involving double-GT and Rag knockout mice as recipients. Lewis rat hearts transplanted into these double-knockout mice survive for > 50 days, suggesting that NK cells and macrophages cannot elicit acute xenograft rejection in the absence of T and/or B cells. We then passively infused anti-Gal IgG1 or IgG3 mAbs to determine their abilites to cause tissue damage and xenograft rejection in vivo. As expected anti-Gal IgG3 infusions of 0.5-0.25 mg/mouse, i.v., resulted in hyperacute rejection in <1 h, while 0.125 mg/mouse dose resulted in rejection in 3-4 h. The histology indicated classic features of hyperacute rejection of platelet microthrombi, C3 and C5 deposition. Surprisingly, anti-Gal IgG3 (0.5-0.25 mg/mouse) was able also to elicit hyperacute rejection and xenograft hearts completely stopped beating in about 2 h. However lower doses of IgG1, 0.125 mg/mouse for 7 daily doses from day 0, could not cause graft rejection. This suggested that despite the higher affinity, anti-Gal IgG1 was less effective at eliciting xenograft rejection. Examination of the rejecting revealed strong C3, but minimal C5 deposition, and platelet microthrombi 15-30 min after anti-Gal Ab infusion. The levels of C3 deposition and platetet microthrombi were significantly reduced at 2 h after anti-Gal Ab infusion. In contrast to IgG3-mediated rejection which was only inhibited by CVF, IgG1-mediated rejection was inhibited by CVF as well as anti-asialo GM1 Abs. These data collectively suggest that IgG3-mediated hyperacute rejection is mediated by the classical complement pathway, while IgG1-mediated hyperacute rejection may be dependent on C3-enhanced ADCC.