The Regulating Role Of S1PR5 Expressed On NK Cells In The Pathogenesis Of Graft Versus Host Disease

Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is widely used in the treatment of hematological disorders and some kinds of solid tumors. Acute graft versus host disease (aGVHD) remains a major cause of morbidity and mortality following transplantation. NK cells are the first recovery immune cells during the immunologic reconstitution after allo-HSCT. Current studies have demonstrated that NK cells could suppress the occurrence of aGVHD without affecting the graft-versus-tumor effects. Sphingosine-1-phosphate receptor 5 (S1PR5) is one kind of G protein-coupled receptors expressed on the surface of NK cells. S1PR5 mediates the migrating process of NK cells from bone marrow and lymphnodes into other tissues, thus exerting its immunological function. Our previous study found that the occurrence of aGVHD in patients following allo-HSCT correlates with the expression level of S1PR5 on NK cells.Objective:This study aimed to explore the regulating role of S1PR5 expressed on NK cells in the pathogenesis of aGVHD.Methods and Results:① The RNA and protein level of S1PR5 from S1PR5 knockout (S1PR5-/-) mice, which was construted by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat and cas9 protein) technology, was detected by qPCR and Western blot, respectively. The result showed that S1PR5 was completely knocked out.② Flow cytometry measured lymphocyte subsets of wild type C57BL/6 mice or S1PR5-/-C57BL/6 mice from different anatomic sites (bone marrow, spleen, mesenteric lymphnodes and peripheral blood) respectively. The percentages of NK cells in bone marrow and lymphnodes of S1PR5-/-C57BL/6 mice were higher than that of wild type C57BL/6 mice. The percentages of NK cells in spleen and peripheral blood of S1PR5-/-C57BL/6 mice were lower than that of wild type C57BL/6 mice.③Acute GVHD model was established by infusing bone marrow cells and spleen cells from wild type C57BL/6 mice (control group) or S1PR5-/-C57BL/6 mice (experimental group) into lethally irradiated recipient BALB/c mice through the tail vein. Then we monitored body weight, aGVHD score and survival rate of mice between these two different groups. We found that mice receiving cells from S1PR5-/-mice survived shorter (P= 0.019) and had higher aGVHD scores (P=0.010) than that in the control group after following up for 2 months post transplantation. However, No significant difference in body weight between these two groups was noted.④ Lymphocyte subsets from different anatomic sites (bone marrow, spleen, mesenteric lymphnodes and peripheral blood) were measured by flow cytometry on the 4th day post transplantation. The percentages of NK cells in bone marrow of mice in the experimental group were higher than that in the control group (P= 0.036). No significant difference in the percentage of NK cells in the spleen, lymphnodes and peripheral blood between these two groups was noted. The percentage of T cells in bone marrow of the experimental group were lower than that in the control group (P=0.023). But there was no difference in the percentage of T cells in the spleen, lymph nodes and peripheral blood between these two groups.⑤ Lymphocyte subsets from different anatomic sites (bone marrow, spleen, mMesenteric lymph nodes and peripheral blood) were measured by flow cytometry on the 30th day post transplantation. The percentages of NK cells in bone marrow of mice of the experimental group were also higher than that in the control group (P-0.037). No significant difference in the percentage of NK cells in the spleen, lymphnodes and peripheral blood between these two groups was noted. The percentage of T cells in the peripheral blood of the experimental group were higher than that in the control group (P=0.031). But there was no difference in the percentage of T cells in the bone marrow, spleen and lymph nodes between these two groups.Conclusions:① CRISPR/Cas9 technology is an effective and efficient method to establish gene knockout mice.② S1PR5 has an effect on NK cells distribution in mice;③ Expressing deletion of S1PR5 in the donor mice can aggravate the severity of aGVHD of the recipient mice;④ Expressing deletion of S1PR5 may inhibit the migrating process of NK cells from the bone marrow, thus decreasing the number of NK cell in the peripheral blood. And this can reduce the protecting effect of NK cells on aGVHD, which can partially explain why expressing deletion of S1PR5 in the donor mice can aggravate the severity of aGVHD in the recipient mice.