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.

Purpose of Review-Anti-non-gal xenoantibodies are a major barrier to the survival of genetically modified porcine xenografts. This review summarizes new information on the contribution of antinon-gal xenoantibodies to the activation of porcine endothelial cells and the pathology of rejection and further provides an update on recent advancements in defining the unique features of anti-nongal xenoantibody structure.

2 3 7 WEDNESDAY Supplement to Transplantation July 27, 2008, Volume 86 Number 2Sproperties. Thus, the expression of hHO-1 on porcine endothelial cells could have benefi cial effects on organ survival after xenotransplantation and could possibly interfere with XAC. In this study we analyzed porcine cells and kidneys expressing the human complement regulator CD55 and the cell protective protein hHO-1. Methods: The protective effects of the transgenes against complementmediated attack were assessed by cytotoxity assays. Organ survival and XAC following the contact of human blood with porcine endothelium, were determined by using an ex-vivo perfusion circuit based on low dose heparin mediated anticoagulation. Porcine kidneys were recovered following in situ cold perfusion with HTK organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled human AB blood. Results: Porcine fi broblast transgenic for CD55/hHO-1 showed increased resistance (up to 40%) against complement mediated attack than fi broblasts from wildtype pigs. Survival of wildtype pig kidneys during organ perfusion with human blood and addition of heparin was 42 +/-26 min. Application of soluble complement inhibition (C1-Inhibitor) prolonged organ survival to 126 +/-72 min. XAC was observed with signifi cantly elevated concentrations of d-Dimer and thrombin antithrombin complex (TAT) combined with consumption of fi brinogen and antithrombin. The histological analyses revealed numerous microthrombi. In contrast, the perfusion of the CD55/hHO-1 transgenic porcine kidneys was feasible for more than 240 min in all perfusion experiments (without C1-Inhibitor). Although elevated levels of d-Dimer and TAT were measured, no signifi cant consumption of fi brinogen and antithrombin occurred. In addition, no microthrombi were detectable histologically. Conclusion: We conclude that the expression of CD55 and hHO-1 in porcine kidneys can prolong organ survival and is able to interfere with XAC in this ex vivo perfusion model. These results suggest that the expression of hHO-1 in combination with CD55 could play an important role in xenograft protection. -defi cient (GalTKO) pig organs were developed to provide a solution to the shortage of human organ donors. These organs do not initiate hyperacute rejection but undergo acute humoral rejection mediated by xenoantibodies directed at non-gal xenoantigens. The structure, specifi city and germline origin of these antibodies has not been determined. Methods: Three rhesus monkeys were immunized with sixty million GalTKO pig endothelial cells in order to induce high levels of xenoantibodies directed at GalTKO xenoantigens. Pre-existing levels of anti-non-gal xenoantibodies were signifi cantly lower than anti-gal natural antibodies in rhesus monkeys. A strong immune response directed at GalTKO xenoantigens was confi rmed by fl ow cytometry after immunization. Pre and post transplant peripheral blood B cells were used to prepare IgG and IgM cDNA libraries for identifi cation o...