Journal of Clinical & Experimental Nephrology

HLA-class Ib expression suppresses neutrophil xenogeneic immune responses against pig cells

Purpose: The expression of Human Leukocyte Antigen (HLA) class â?  molecules regulates innate immune responses of Natural Killer (NK) cells and macrophages. In this study, we investigated the regulatory responses of Human Leukocyte Antigen G1(HLA-G1) and Human Leukocyte Antigen E (HLA-E) on human neutrophils.

Material and methods: The expression of counter receptors on neutrophils was detected using flow cytometry. Complementary DNA (cDNA) of HLA-G1 and HLA-E were introduced into Swine Endothelial Cells (SECs) to establish SEC/HLA-G1 and SEC/HLA-E cell lines, respectively. These cell lines were then co-cultured with neutrophils to assess cytotoxicity. Subsequently, Reactive Oxygen Species (ROS) production levels were calculated using Cell Reactive Oxygen Green (CellROX Green). Neutrophil Extracellular Traps (NETosis) in neutrophils of SEC cells was measured using SYTOX Green.

Results: The expression of Natural Killer Group 2 member A (NKG2A), Immunoglobulin-Like Transcript 2 (ILT-2) and ILT-4 on neutrophils was <20%, approximately 30% and approximately 65%, respectively. No significant differences in cytotoxicity were observed in SEC/HLA-E and wild-type SEC cells, whereas SEC/HLA-G1 cells exhibited significant reduction of approximately 35% and 25% in the cytotoxicity after 4 h of incubation with Phorbol 12-Myristate 13-Acetate (PMA) and 24 h of incubation without PMA, respectively. Additionally, SEC/HLA-G1 cells significantly inhibited ROS production. HLA-G1 also suppresses NETosis induction in neutrophils.

Conclusion: These results indicate that HLA-G1 effectively regulates the xenogeneic immune responses of neutrophils against SECs in xenotransplantation.

Purpose: The expression of Human Leukocyte Antigen (HLA) class â?  molecules regulates innate immune responses of Natural Killer (NK) cells and macrophages. In this study, we investigated the regulatory responses of Human Leukocyte Antigen G1(HLA-G1) and Human Leukocyte Antigen E (HLA-E) on human neutrophils.

Material and methods: The expression of counter receptors on neutrophils was detected using flow cytometry. Complementary DNA (cDNA) of HLA-G1 and HLA-E were introduced into Swine Endothelial Cells (SECs) to establish SEC/HLA-G1 and SEC/HLA-E cell lines, respectively. These cell lines were then co-cultured with neutrophils to assess cytotoxicity. Subsequently, Reactive Oxygen Species (ROS) production levels were calculated using Cell Reactive Oxygen Green (CellROX Green). Neutrophil Extracellular Traps (NETosis) in neutrophils of SEC cells was measured using SYTOX Green.

Results: The expression of Natural Killer Group 2 member A (NKG2A), Immunoglobulin-Like Transcript 2 (ILT-2) and ILT-4 on neutrophils was <20%, approximately 30% and approximately 65%, respectively. No significant differences in cytotoxicity were observed in SEC/HLA-E and wild-type SEC cells, whereas SEC/HLA-G1 cells exhibited significant reduction of approximately 35% and 25% in the cytotoxicity after 4 h of incubation with Phorbol 12-Myristate 13-Acetate (PMA) and 24 h of incubation without PMA, respectively. Additionally, SEC/HLA-G1 cells significantly inhibited ROS production. HLA-G1 also suppresses NETosis induction in neutrophils.

Conclusion: These results indicate that HLA-G1 effectively regulates the xenogeneic immune responses of neutrophils against SECs in xenotransplantation.

Author(s): Koki Takase1*, Katarzyna Gadomska1, Akira Maeda1, Jun Matsui1, Kengo Nakahata1, Motonari Nomura1, Masafumi Kamiyama1, Takehisa1, Ueno1, Shunsuke Saito2, Akihiro Ike3, Soichi Matsumura4, Yoichi Kakuta4, Hiroshi Eguchi1, Hiroomi Okuyama1 and Shuji Miyagawa1

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