Role of complement regulatory protein expression and CD154 blockade in Gal‐independent xenograft rejection

Abstract

IntroductionHyperacute (HAR) and delayed xenograft (DXR) rejection are well defined barriers following discordant transplantation of porcine organs into primate recipients. Significant progress has been made in recent years to overcome these immunological barriers, based on the development of pigs genetically modified to reduce immunogenicity: [1,2] A major step has been the generation of pigs lacking expression of Gala1,3Gal (GalTKO), the principal carbohydrate target of human anti‐pig antibody. [3,4] However, both in vitro and in vivo studies demonstrated that non‐gal antibodies have the ability to activate endothelial cells, and thus, amplify procoagulant molecular incompatibilities between pig and primate.[5,6] Based on our prior work with pigs expressing the Gal epitope, we tested the hypothesis that over‐expression of human complement regulatory proteins (hCRP) and/or blockade of the CD154/CD40 costimulation pathway will result in reduced graft injury, anti‐pig responses and coagulation dysregulation. Briefly, twenty two organs (13 hearts, 9 kidneys) from GalT‐KO pigs (9 expressing hCRP) were transplanted into baboons that received no immunosuppressive therapy, or a therapeutic regimen based on costimulation blockade. The incidence of HAR was decreased in GalTKO organs expressing hCRP as compared to GalTKO. Pre‐transplant anti‐non‐Gal antibody titers were inconsistently associated with early GalT‐KO xenograft failure. In contrast, coagulation cascade activation and platelet activation correlated closely with incidence of HAR of GalTKO organs. Strong elicited anti‐non‐gal antibody responses were detected after transplantation of a GalTKO organ. Delayed xenograft rejection was associated with coagulation dysregulation and elicited anti‐non‐gal antibody production. Future work in xenotransplantation should place emphasis on further targeting of anti‐non gal humoral immune responses and inhibiting coagulation activation. References 1. Pierson RN III. JAMA 2009; Current status of xenotransplantation. 30: 967–969. 2. Cooper DK, Dorling A, Pierson RN III, et al. Alpha1,3‐galactosyltransferase gene‐knockout pigs for xenotransplantation: where do we go from here? Transplantation 2007; 84: 1–7. 3. Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3‐galactosyltransferase gene‐knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 2005; 11: 32–34. 4. Kuwaki K, Tseng YL, Dor FJ, et al. Heart transplantation in baboons using alpha1,3‐galactosyltransferase gene‐knockout pigs as donors: initial experience. Nat Med 2005; 11: 29–31. 5 Saethre M, Baumann BC, Fung M, Seebach JD, Mollnes TE. Characterization of natural human anti‐non‐gal antibodies and their effect on activation of porcine gal‐deficient endothelial cells. Transplantation. 2007; 84: 244–250. 6. Hisashi Y, Yamada K, Kuwaki K, et al. Rejection of cardiac xenografts transplanted from alpha1,3‐galactosyltransferase gene‐knockout (GalT‐KO) pigs to baboons. Am J ...

Development of a consensus protocol to quantify primate anti‐non‐Gal xenoreactive antibodies using pig aortic endothelial cells

“…In summary, through a collaborative consortium effort, we have developed a consensus protocol to detect antibodies against non‐Gal pig antigens to facilitate interlaboratory comparisons, and evaluated this approach using sera from baboons sensitized by GalKO cardiac xenografts. Whether the assay is suitable for xenotransplantation of other organs or pancreatic islets is not clear at this time, albeit our experience with kidney xenografts in baboons and islet xenografts in rhesus macaques suggests that the assay is informative in those contexts as well. We offer recommendations on procedures and reagents and discuss sources of assay variability that should aid in the reporting of humoral immune responses to non‐Gal epitopes.…”

“…The recent development of pigs in which the α‐galactosyl transferase gene has been deleted (GalTKO) and human complement regulatory proteins have been over‐expressed has further reduced but not eliminated the incidence of hyperacute rejection . Xenotransplantation using GalTKO organs has eliminated a role for anti‐Gal antibody in xenograft rejection but has not eliminated antibody‐mediated rejection overall as both preformed and induced antibodies binding to GalTKO organs (i.e.…”

Minimal effect of bortezomib in reducing anti‐pig antibodies in human leukocyte antigen‐sensitized patients: a pilot study

“…The targets for these anti-non-Gal antibodies remain largely unknown [43]. Although their effect is to some extent abrogated by expression of one or more human complement-regulatory proteins on the pig vascular endothelium [44][45][46][47], this is not entirely protective, particularly in regard to vascular endothelial cell activation, which may be an initiating factor in the development of coagulation dysregulation [48][49][50][51][52][53][54][55]. This may take the form of the development of a thrombotic microangiopathy or a consumptive coagulopathy.…”