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Novel experimental models to test human NK cell recruitment to porcine endothelium and their role in cellular xenograft rejection

Amandine Pradier¹, Ruhollah Heyrani Nobari¹, Anne-Laure Millard², Anjan Bongoni³, Mihai Constantinescu⁴, David Kiermeir⁴, Mårten Schneider⁵, Andrea Bähr⁶, Nikolai Klymiuk⁶, Robert Rieben³, Jörg D. Seebach¹

¹Department of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland; ²Division of Infectious Diseases, University Hospital, Zurich, Switzerland; ³Department of Clinical Research, University of Bern, Bern, Switzerland; ⁴Clinic of Plastic and Hand Surgery, University Hospital, Bern, Switzerland; ⁵Laboratory of Vascular Immunology, University Hospital, Zurich, Switzerland; ⁶Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany

In vitro, interactions between human NK cells and porcine endothelial cells (pEC) are characterized by NK cell recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity under static conditions was achieved by the expression of HLA-E on pEC and blocking of activating NK receptors. However, this strategy has not yet been tested under physiological shear stress or in vivo/ex vivo. Therefore, the aims of this study were to evaluate whether human NK cells damage pEC in a flow system and to study NK cell recruitment and infiltration of porcine tissues using an ex vivo perfusion system of HLA-E transgenic pig legs with human blood.

Monolayers of pEC were perfused in vitro with human NK cells in a flow chamber for several hours. NK cells adhesion and transmigration was observed from the beginning, however, damage of the layer was noted only after 6h of perfusion. Two wild type and 2 HLA-E/CD46 transgenic pig legs were perfused ex vivo with heparinized human blood. Blood samples were collected at several time points and PBMC were analyzed by FACS. A strong diminution of NK cells was observed after 7h 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 was observed.

These results confirmed previous in vitro studies demonstrating that pEC damage was mediated by human NK cells. Moreover, our preliminary results showed that the expression of HLA-E/CD46 provided a partial protection with regard to NK cell recruitment and tissue infiltration. In conclusion, these systems represent powerful tools to study the basic molecular mechanisms taking place in a setting of xenotransplantation and to evaluate the protection from early cell-mediated rejection mechanisms in tissues originating from transgenic pigs.