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P-21.14 CD177 on swine cells suppresses xenogeneic macrophage-mediated cytotoxicity

Pei-Chi Lo, Taiwan

undergraduate student
pediatric surgical
Osaka university

Abstract

CD177 on swine cells suppresses xenogeneic macrophage-mediated cytotoxicity

Pei-Chi Lo1, Akira Maeda1, Tomohisa Yoneyama1, Chiyoshi Toyama1, Hiroshi Eguchi1, Katsuyoshi Matsunami2, Yuko Tazake1, Hiroomi Okuyama1, Shuji Miyagawa1,3.

1Pediatric Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan; 2Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; 3 International Institute for Bio-Resource Research, Meiji University, Tokyo, Japan

Introduction: Xenotransplantation is one of the strategies to overcome the problems of organ shortage in the transplant. However, innate cellular rejections by NK cells, macrophages and neutrophils cause severe rejection in xenotransplantation. In our previous study shown CD31-CD31 binding induces immunoreceptor tyrosine-based inhibitory motif (ITIM) activation of neutrophil and suppresses neutrophil-mediated cytotoxicity. Yet, it fails to suppress macrophage-mediated cytotoxicity. Compare to CD31, CD177, a glycosyl-phosphatidylinositol-linked N-glycosylated cell surface glycoprotein, shows a higher affinity to bind to CD31. Therefore, we expect CD177-CD31 binding substitutes CD31-CD31 binding to suppress both macrophage- and neutrophil-mediated cytotoxicity.
Materials and Methods: A plasmid PCXN2-β-actin promoter-human CD177 was introduced into swine endothelial cells (SEC) by lipofection. CD177-transfected SEC clones were isolated by limiting dilution. The expression of CD177 on the SEC transfectant was confirmed by flow cytometry. A human monocyte-like cell line, THP-1 was activated as a macrophage-liked cell with 200 nM phorbol 12-myristate 13-acetate (PMA) for 1 day. Peripheral blood monocytes (PBMCs) were obtained by density gradient centrifugation. CD14+ PBMCs were isolated by magnetic selection and were cultured with 100 ng/ml GM-CSF for 7 days maturation. The expression of CD31 on macrophages and PMA-THP1 was confirmed by flow cytometry. To evaluate the role of human CD177 in xenogeneic macrophage-mediated cytotoxicity, SEC or SEC/CD177 was co-cultured with PMA-THP-1  or macrophages for 24 hours. The cytotoxicity of immune cells to the SEC was measured by a WST-8 assay. 
Results and Discussion: High CD177 expression SEC clones were isolated successfully. CD177 but not CD31 demonstrated significant suppression of THP-1-induced cytotoxicity(%Cytotoxicity. SEC: 40.5±1.8%, SEC/CD31: 41.2±2.8%, SEC/CD177: 29.7±2.9%, n=6, p<0.05). Similar results also presented on the macrophage-mediated cytotoxicity(%Cytotoxicity. SEC: 32.7±2.5%, SEC/CD31: 33.8±2.7%, SEC/CD177: 25±1.9%, n=10, p<0.01).
Conclusion: These findings suggest that human CD177 expression on swine cells suppresses xenogeneic macrophage-mediated cytotoxicity.  CD177 will be the next candidate to control the xenogeneic macrophage-mediated rejection.

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