The Anti-Non-Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

Abstract

Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into non-human primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgVH genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3-21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of “anti-non-gal” xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies crossreact with carbohydrate xenoantigens. The galactosyltransferase 2 gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgVH genes that are restricted to IGHV3-21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

Cardiac xenotransplantation

“…((10) and GWB personal comm.) Comparisons of neutral and acidic glycolipids from Gal-positive and GTKO pig organs found no evidence of αGal in GTKO tissues (11) and molecular analysis failed to observe expression of the iGb3 α-galactosyltransferase in GTKO pig cells (12). Xenotransplantation of GTKO hearts has no effect on preformed anti-Gal antibody and does not stimulate an anti-Gal antibody response (13, **14).…”

The anti-nonGal xenoantibody response to α1,3-galactosyltransferase gene knockout pig xenografts

“…It has been postulated that anti-non-gal xenoantibodies may bind to a residual gal carbohydrate expressed on GalTKO pig cells as a consequence of iGb3 synthase expression. We examined whether iGb3 synthase might be expressed in GalTKO pig cells using primers designed to identify conserved regions in the galactosyltransferase 2 gene (A3galt2 or iGb3 synthase) in humans, rats, and mice [11 * ]. Our data confirmed that expression of the gene encoding this transferase is detected in GalTKO mice but we did not detect expression of this gene in GalTKO pig cells, GalTKO fetal pig fibroblasts or in minipig kidney cells (MPK) from wild type pigs.…”

“…There is now evidence from our laboratory and others that GalT-KO xenoantibodies pre-exist in humans and non-human primates at significantly lower levels than anti-Gal xenoantibodies [11 * –14]. Since a threshold level of xenoreactive antibodies is necessary to initiate xenograft rejection [19], it is not surprising that hyperacute rejection of GalTKO xenografts has not been reported.…”

“…These IgM anti-non-gal xenoantibodies may be responsible for initiating xenograft rejection when they reach sufficient levels after placement of the xenograft. We found that in naive rhesus monkeys, pre-existing IgM xenoantibodies bound to an average of 8% of GalT-KO pig cells, considerably lower than the 78–99% binding of pre-existing IgM xenoantibodies to wild-type pig cells that express the gal carbohydrate [11 * ]. Interestingly, cytotoxic natural anti-non-Gal xenoantibodies are either absent or present in very low levels in infant baboons and humans [12].…”

“…Pre-existing levels of anti-non-gal xenoantibodies have recently been shown to be low in humans and in non-human primates [11 * –13], and the induced xenoantibody response following exposure to GalTKO pig endothelial cell xenoantigens is now known to be restricted [11 * ]. The nature of the target xenoantigen(s), however, still remains elusive and conventional immunosuppressive drugs do not effectively prevent xenoantibody-mediated rejection.…”