Functional evaluation of hTM, CD46 and HLA‐E transgenes in vitro and in pig leg xenoperfusion experiments. (March 2014) - Bongoni, Anjan K.
Abstract Background Besides α1, 3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig‐to‐human xenograft rejection are being investigated. hCD46/HLA‐E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig‐to‐human xenoperfusion model. In addition, expression of human thrombomodulin (hTM‐) on wild‐type and/or multi‐transgenic (GalTKO/hCD46) background was evaluated to overcome pig‐to‐human coagulation incompatibility. Methods hCD46/HLA‐E double transgenic as well as wild‐type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E‐selectin and VCAM‐1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM‐only and multi‐transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti‐coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL‐8 (P = 0.019) as well as human C3a, C5a and soluble C5b‐9 were significantly (P < 0.05–<0.0001) lower in blood perfused through hCD46/HLA‐E transgenic as compared to wild‐type limbs. C3b/c, C4b/c, and C6 deposition as well as E‐selectin and VCAM‐1 expression were significantly (P < 0.0001) higher in tissue of wild‐type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA‐E is associated with a reduction of NK cell tissue infiltration (P < 0.05). A rapid decrease of platelets was observed in all xenoperfusions. In vitro findings showed that PAEC express ASGR1 and suggest that this molecule is involved in human platelet phagocytosis. In vitro, we found that the amount of APC in the supernatant of hTM transgenic cells increased significantly (P < 0.0001) with protein C concentration in a dose‐dependent manner as compared to control PAEC lacking hTM, where the turnover of the protein C remained at the basal level for all of the examined concentration. In further experiments, hTM also showed the ability to prevent blood coagulation by three‐ to four‐fold increased (P < 0.001) clotting time as compared to wild‐type PAEC. The formation of TAT complexes was significantly lower when hTM‐transgenic cells (P < 0.0001) were used as compared to wild‐type cells. Conclusions Transgenic hCD46/HLA‐E expression clearly reduced humoral xenoresponses since the terminal pathway of complement, endothelial cell activation, inflammatory cytokine production and NK‐cell tissue infiltration were all down‐regulated. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human platelets during pig‐to‐human xenotransplantation. In addition, use of the hTM transgene has the potential to overcome coagulation incompatibilities in pig‐to‐human xenotransplantation.