Introduction: Cellular xenogeneic rejection by the innate immune system is one of the most important immunological obstructions after the alpha-Gal-knocked out era. We previously demonstrated that hCD31 on swine endothelial cell (SEC) suppresses the xenogeneic reaction by neutrophil. This suppression is induced by the inhibitory signals from ITIM via a homophilic binding of hCD31. Since CD31 is expressed on a variety of cells, we hypothesized that CD31 could be also available to suppress NK cytotoxicity. In this study, the suppressive effect of hCD31 in NK cell-mediated xenogeneic rejection was investigated.

Materials and Methods: cDNAs for hCD31 was cloned into the pCXN2L (β-actin promotor + CMV enhancer + neomycin resistance) expression vector. Next, the plasmids were introduced into the SEC line, MYP30, by lipid-mediated DNA transfection methods. Through drug resistance selection, SEC/hCD31 was established as a monoclonal clone. The expression of hCD31 on the SEC/hCD31 and peripheral blood NK cells was checked by flow cytometry.

To verify our hypothesis, SEC and SEC/hCD31 were then co-cultured with peripheral blood mononuclear cells (PBMCs) for 4 hours, and the cytotoxicity was evaluated with WST-8 assay. Next, CD107a (lysosomal-associated membrane protein 1: LAMP-1) expression was assessed by flow cytometry to study the degranulation of NK cells. PBMCs were collected after co-culture with SEC and SEC/hCD31 for 4 hours and stained with an anti-CD107a antibody, followed by the flow cytometry.

Results: CD31 was found to be expressed on more than 95% of peripheral blood NK cells.  hCD31 on SECs significantly suppressed the NK cell-mediated cytotoxicity compared to naive SEC (45.2% vs. 36.8%, p=0.0362, N=15 and 41.9% vs. 31.6%, p=0.0231, N=14, respectively).  In the degranulation assay using an anti-CD107a antibody, hCD31 showed a significant suppression on NK cell degranulation in both MFI (113.6 vs. 39.8, p=0.0189, N=5) and CD107a positive cell rate (4.284% vs. 1.476%, p=0.002, N=5) compared to naive SEC.

Conclusion: These findings suggest that hCD31 is effective to suppress the xenogeneic rejection by NK cell.  In the future, it is necessary to conduct a comparative study with the inhibitory effects by HLA-E and G1.