Transgenic strategies to overcome cell‐mediated and acute vascular rejection of pig‐to‐human xenografts
Authors: Klymiuk N. , Keßler B. , Kurome M. , Wuensch A. , Wolf E. 
Published: April 2010

The development of α(1,3)‐galactosyltransferase deficient pigs along with overexpression of complement regulatory proteins was an important step to overcome hyperacute rejection of pig organs transplanted into primates. However, the lack of galactose‐α(1,3)‐galactose (αGal) epitopes does not protect against subsequent rejection mechanisms, such as acute vascular rejection and rejection by immune cells which play crucial roles to achieve prolonged graft survival in pig‐to‐human xenotransplantation models. Our task within the DFG Transregio Research Unit “Xenotransplantation” is primarily the development of transgenic strategies to overcome cell mediated rejection of pig‐to‐primate xenografts. Our first target was the lysis of porcine cells by activated human natural killer (NK) cells, which involves antibody‐dependent and ‐independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically HLA‐E, a trimeric complex consisting of the HLA‐E heavy chain, β2‐microglobulin (β2m) and a peptide derived from the leader sequence of some MHC class I molecules. To use this mechanism for protection of pig tissues against human NK cell‐mediated cytotoxicity, we generated transgenic pigs carrying genomic fragments of HLA‐E with a HLA‐B7 signal sequence and of human (hu) β2m. Immunohistochemistry revealed the presence of HLA‐E and huβ2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA‐E/huβ2m transgenic pigs are effectively protected against human NK cell‐mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA‐E/huβ2m expression on porcine endothelial cells inhibited the secretion of IFN‐γ by co‐cultured human NK cells [1]. Currently the HLA‐E/huβ2m transgenes are crossed on a CD46 transgenic background to facilitate evaluation of the NK cell inhibitory effect in ex vivo perfusion studies of multitransgenic pig hearts with human blood.The second target is inhibition of T cell activation by expression of CTLA‐4Ig. This recombinant soluble fusion protein binds to CD80 and CD86, blocking their interaction with CD28 and thereby inhibiting T cell‐dependent antibody production and T cell proliferation. The survival of human, rabbit and porcine islets after transplantation into diabetic mice was found to be prolonged after treatment with CTLA‐4Ig. As a transgenic strategy to protect porcine islets against human T cells, we cloned an expression vector, in which the coding sequence for LEA29Y, a more potent variant of CTLA‐4Ig, is placed under the transcriptional control of the porcine INS promoter. This expression cassette is linked to a floxed neomycin resistance cassette in order to facilitate selection of stably transfected fetal fibroblasts (FF). After two passages under selection, pooled FF colonies were used for nuclear transfer. Cloned embryos were laparoscopically transferred to recipient gilts, resulting in two pregnancies and eight offspring, seven of which were transgenic. Immunohistochemistry of pancreas sections from one selected founder demonstrated the presence of LEA29Y in the pancreatic islets, the expression pattern being in agreement with β‐cell specific expression. Protection of INS‐LEA29Y transgenic islets will be tested in diabetic mouse models with a humanized immune system.In addition to cell mediated rejection mechanisms we attempted to target acute vascular rejection mechanisms by expression of human thrombomodulin (hTM) on porcine endothelial cells. This has been discussed as an important step to overcome incompatibilities in the regulation of human blood coagulation after perfusion of porcine blood vessels. To achieve efficient expression of hTM we constructed three different expression vectors: (i) the hTM gene with homologous 5′ sequences and polyadenylation signal; (ii) the hTM gene driven by the porcine TM promoter, but with homologous 3′ flanking sequences; and (iii) the hTM gene with the porcine TM promoter and a polyadenylation signal and 3′ flanking sequences from the bovine growth hormone gene. The latter expression vector was linked to the neomycin resistance cassette mentioned above and transfected into fetal fibroblasts. A pool of stably transfected cell clones was used for nuclear transfer. Transfer of cloned embryos to recipients resulted in three pregnancies and eight piglets, all of which were transgenic. Five of six founders investigated showed strong expression of hTM on endothelial cells in heart and kidney. Cells from selected animals are currently being used for re‐cloning to provide transgenic pig organs for ex vivo perfusion studies with human blood to evaluate the efficacy of hTM expression.Supported by the DFG (FOR 535 “Xenotransplantation”).Reference1. Weiss, et al. HLA‐E/human beta2‐microglobulin transgenic pigs: protection against xenogeneic human anti‐pig natural killer cell cytotoxicity. Transplantation 2009; 87: 35–43.