Introduction: It is well known that the immune response of NK cells is regulated by their HLA class I expression on target cells, and this is also well known in the field of xenotransplantation. Furthermore, previous studies revealed that the immune response of macrophages against porcine endothelial cells (SECs) could be modulated by expressing HLA class Ib on porcine endothelial cells (SECs). In this study, we investigated to what extent the expression of HLA class Ib (HLA-E and HLA-G1) on target cells influences the immune response of human neutrophils.
Method: First, the cDNAs of HLA-E and HLA-G1 were inserted into an expression vector (pCAGGS), and then introduced into SEC to establish SEC/HLA-E and SEC/HLA-G1 lines. Neutrophils were isolated from peripheral blood. We then examined the expression of their counterreceptors NKG2A, ILT-2, and ILT-4 on neutrophils using a flow cytometer. These cells (SEC/HLA-E and SEC/HLA-G1) were co-cultured with human neutrophils in the presence or absence of PMA to assess cytotoxicity. Additionally, the ROS generation level was calculated by CellROX Green Reagent. The NETosis response of neutrophils to SEC cells was also evaluated using SYTOX Green.
Result: The expression of NKG2A in human neutrophils was less than 20%, whereas ILT-2 was about 30%, increasing to 40% upon PMA stimulation, and ILT-4 was about 65%. Correspondingly, no significant difference in cytotoxicity rate was observed with SEC/HLA-E compared to wild-type SEC, regardless of the presence or absence of PMA stimulation. On the other hand, SEC/HLA-G1 was significantly suppressed by approximately 35% with PMA stimulation (4 hours) and by approximately 25% without PMA stimulation (24 hours). Similarly, SEC/HLA-G1 showed significant inhibition of ROS production. Furthermore, HLA-G1 showed the ability to suppress the induction of NETosis in neutrophils.
Conclusion: These results suggest that the expression of HLA-G1 on SEC can significantly suppress the xenogeneic immune responses of neutrophils to SEC, indicating the effectiveness of HLA-G1 gene transfer in the field of xenotransplantation.