Use of porcine tumor necrosis factor receptor 1-Ig fusion protein to prolong xenograft survival.
BACKGROUND
Delayed rejection of xenografts is a major hurdle that needs to be addressed to achieve long-term engraftment in the pig-to-primate transplant setting. Both vascular and avascular xenografts are susceptible to a delayed rejection process that comprises humoral and cellular responses. Tumor necrosis factor (TNF) is believed to play a role in this process by promoting cell activation, apoptosis and the recruitment of inflammatory cells. To address this problem, we engineered the donor cell in such a way that it could block both human and porcine TNF.


METHODS
We produced a recombinant fusion protein containing the extracellular domain of the porcine TNF-Receptor 1 and an IgG Fc moiety (pTNFR1Ig). We first evaluated by flow cytometry the pTNFR1Ig capacity to prevent TNF alpha-induced expression of SLAI, SLAII, VCAM-1, ICAM-1 and E-selectin on the cell surface of porcine aortic endothelial cells (PAEC). The effect on TNF alpha-mediated cell death was also assessed by propidium iodide staining after incubating PAEC with TNF alpha plus cycloheximide for 24 h. PAEC and porcine fibroblasts were subsequently engineered by retroviral infection to express and secrete pTNFR1Ig and their resistance to the TNF alpha effects was tested in vitro. Finally, we transplanted mock-control and pTNFR1Ig-expressing PAEC under the kidney capsule of BALB/c mice in the absence of immunosuppression and examined the degree of rejection at 2 and 3 weeks post-transplantation.


RESULTS
Treatment with pTNFR1Ig resulted in a very potent blockade of human, porcine and murine TNF alpha activity on porcine cells. It inhibited the upregulation of all cell surface markers of activation tested as well as the TNF alpha-mediated cell death. Moreover, pTNFR1Ig-expressing PAEC showed prolonged engraftment in a pig-to-mouse xenotransplant model.


CONCLUSIONS
Incorporation of strategies that block TNF may prove useful in the development of xenografts resistant to delayed rejection.
Department of Molecular and Preclinical Sciences, Alexion Pharmaceuticals Inc, Cheshire, CT, USA. ccosta@iro.es
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D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D006084:Graft Rejection / Q000188:drug therapy / Q000517:prevention & control*; D006085:Graft Survival / Q000187:drug effects*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism / Q000494:pharmacology*; D007142:Immunoglobulin gamma-Chains; D016030:Kidney Transplantation; D051379:Mice; D008807:Mice, Inbred BALB C; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine; D014183:Transplantation, Heterologous; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism
D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C099374:Ro 45-2081