Ex vivo perfusion of HLA‐E/CD46 transgenic pig limbs with human blood: evaluation of NK cell recruitment

In vitro, interactions between human NK cells and porcine endothelial cells (pEC) are characterized by NK cell recruitment and cytotoxicity. NK cells play a role in allo‐ and xenotransplantation due to the incompatibility of the MHC class I molecules expressed on transplanted cells and tissues on one hand, and the MHC class I specific inhibitory NK receptors of the recipient on the other hand. In this context we published previously that: (i) the expression of human HLA‐E on pEC partially protects from polyclonal huNK cytotoxicity and completely protects from killing by NKG2Abright NK clones in vitro; but does not affect the adhesion of huNK cells to pEC or the heteroconjugate formation between huNK and porcine cells (1, 2); (ii) lymphoblasts and pEC derived from HLA‐E/human beta2microglobulin transgenic pigs are effectively protected against huNK cell‐mediated cytotoxicity and inhibit the secretion of IFN‐gamma by co‐cultured huNK cells in vitro, depending on CD94/NKG2A expression on the NK cells (3); (iii) HLA‐G expression on pEC protects partially against direct huNK cytotoxicity but not against ADCC (4); (iv) HLA‐G expression inhibits rolling adhesion of activated huNK cells on pEC (5, 6); (v) HLA‐Cw3 expression on pEC protects against NK cell‐mediated cytotoxicity (7), HLA‐Cw4 reduced NK cell adhesion and cytotoxicity; HLA‐Cw4 and HLA‐Cw3 co‐expression is not sufficient to completely overcome NK cytotoxicity via recognition of the KIR CD158a (KIR2DL1) and CD158b (KIR2DL2/3) receptors (8). The aim of this study was to evaluate NK cell recruitment and infiltration of porcine tissues using an ex vivo perfusion system of HLA‐E transgenic pig legs with human blood. Over the past year, a pilot study has been set up in order to establish an experimental protocol that would allow for the study and the evaluation of the rejection mechanisms occurring during xenotransplantation and especially following reperfusion of pig organs with human blood (9). Amputated pig legs were perfused with heparinised human blood over a period of 12h. Blood samples were collected at several time points and blood cell populations were analyzed by flow cytometry (FACS) analysis. Two wild type and 2 HLA‐E/CD46 transgenic pig legs were perfused ex vivo. A strong diminution of NK cells was observed after 7 h of perfusion in both the wild type and the transgenic legs. However, this decrease occurred earlier in the wild type leg than in the transgenic. In addition, the apparition of a SLA‐I+ cells population of pig origin of higher granularity than the lymphocyte population over time was observed. We hypothesized that these cells could be pig endothelial cells detached from the vessel wall due to xenorejection, or alternatively originating from the pig bone marrow. These results confirmed previous in vitro studies demonstrating that pEC damage was mediated by human NK cells. Moreover, the expression of HLA‐E/CD46 provided a partial protection with regard to NK cell recruitment and tissue infiltration. From these preliminary experiments the following conclusions were drawn: Pig leg perfusion with human blood was feasible for up to 12 h with stable perfusion parameters thus extending the observation time compared to previously described pig kidney, lung and beating heart perfusion systems. pH and potassium were maintained at normal levels and muscle stimulation resulted in contraction throughout the entire perfusion. Thus the pig leg reperfusion system appears to be a stable and optimized system to study rejection mechanisms over several hours in the pig‐to‐human xenotransplantation setting. This system allows for the study of the dynamics of the different cell populations in the blood. Indeed, a strong diminution of NK, NKT and T cells was observed. In addition, this system enables to study the apparition of cells of pig origin in the circulating blood. In conclusion, this system represents a powerful tool to study the basic molecular mechanisms taking place in a setting of xenotransplantation, and in particular to evaluate the protection from early cell‐mediated rejection mechanisms in tissues originating from transgenic pigs. References:  F orte P , L ilienfeld BG , B aumann BC , S eebach JD . Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol 2005; 175: 5463–5470. 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