The suppression of xenogeneic NK cell degranulation by human CD31
Akira Maeda1, Shuhei Kogata1, Pei-Chi Lo1, Keigo Iemitsu1, Makiko Tani1, Fitri Hasnaulia1, Koki Takase1, Kazunori Masahata1, Masafumi Kamiyama1, Hiroshi Eguchi1, Hiroomi Okuyama1, Shuji Miyagawa1.
1Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Suita, Japan
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.
