Preliminary Study On Siglec-G Deficiency Promoting Negative Immune Regulatory Function Of MDSC

Hematopoietic stem cells(HSCs)can differentiate into common myeloid progenitors(CMP),which in turn differentiate into immature myeloid cells(IMCs).IMCs further differentiate into mature macrophages,dendritic cells or neutrophils.When the body occurs pathological conditions such as tumor,inflammation,infection,trauma and so on,the normal differentiation of IMCs can be blocked by inflammatory factors or tumor-derived cytokines,inducing them to become Myeloid derived suppressor cell(MDSC).They are recruited,amplified,and activated in the peripheral blood,bone marrow,or lesions.It has been reported that G-CSF,IL-6 and CXCR2 promote the expansion of MDSC.M-CSF,IL-13 and IL-33 promoted MDSC recruitment.GM-CSF,IL-2R and IFN-γ promoted the activation of MDSC.MDSC can inhibit the activation of T cells by expressing Arginase 1(Argl),Inducible nitric oxide synthase(iNOS),IL-10 and TGF-β,thereby exerting its immunosuppressive function.The phenotype of MDSC in mice was CD11b+Gr-1+.MDSC could be divided into two subsets:granulocytic(PMN-MDSC,CD11b+Ly6Clow Ly6G+)and monocytic(M-MDSC,CD11b+Ly6Chigh Ly6G-).M-MDSC mainly secrete nitric oxide synthase 1(NOS1),and PMN-MDSC mainly secrete reactive oxygen species(ROS)and Argl to exert their effects.Siglec-G is a member of the sialic acid binding immunoglobulin type lectins(Siglecs)family.Its cytoplasmic tail contains an immunoreceptor tyrosine inhibitory motif,which is expressed on the surface of B cells,DC cells and macrophages.Siglec-G can inhibit the activation,proliferation and survival of B-la cells.Inhibit the production of pro inflammatory cytokines by DC cells and IFN-β by macrophages.It is not clear whether Siglec-G can regulate the function of MDSC.If Siglec-G regulates the function of MDSC,it is not clear whether it has an effect on tumor progression.To explore the effect of Siglec-G deficiency on tumor progression,we first dynamically observed the tumor growth in WT and Siglecg-/-tumor-bearing mice.The results showed that Siglecg-/-tumor-bearing mice had faster tumor growth,heavier tumor weight and larger tumor size.To investigate whether Siglec-G deficiency promotes tumor progression by affecting immune cells,we used WT(wild type,WT)and Siglec-G deficient(Siglecg-/-)mice to generate B16F10 melanoma and MC38 colon cancer xenografts,respectively.The proportion and number of MDSC in the spleen of WT and Siglecg-/-tumor-bearing mice were measured by flow cytometry and fluorescent particle counting,respectively.Compared with WT tumorbearing mice,the proportion and number of MDSC and PMN-MDSC in spleen of Siglecg-/tumor-bearing mice were decreased,but the proportion and number of M-MDSC were not significantly different.To further explore the mechanism of MDSC reduction in the spleen of Siglecg-/-tumor-bearing mice,the proliferation and apoptosis of MDSC in the spleen of WT and Siglecg-/-tumor-bearing mice were detected by flow cytometry.Bone marrow cells from WT and Siglecg-/-mice were induced with GM-CSF in vitro to detect the differentiation ability of MDSC(subsets).The results showed that compared with WT tumor-bearing mice,the proliferation and apoptosis of spleen MDSC in Siglecg-/-tumor-bearing mice were not changed.Bone marrow cells from Siglecg-/-mice had a decreased ability to differentiate into MDSC and PMN-MDSC compared with WT mice.To investigate the functional effects of Siglec-G deficiency on MDSC,we subcutaneously injected B16F10 melanoma cell line into WT and Siglecg-/-mice.Single cell suspension was prepared from mouse spleens 17 days later,and MDSC were sorted and subjected to RNA-SEQ.Analysis of RNA SEQ results suggested that Siglec-G deficiency promoted the expression of characteristic genes related to immunosuppressive function of MDSC.We further compared the differences in enzyme activities and cytokine expression of spleen MDSC between WT and Siglecg-/-tumor-bearing mice at the protein level.Compared with WT tumor-bearing mice,Siglecg-/-tumor-bearing mice showed no change of type ⅠArginase-1(Argl)activity,NOS activity and ROS production in MDSC,but reduced TNF-αsecretion and increased TGF-β and IL-10 secretion.To investigate the effect of Siglecg-/MDSC on the function of activated T cells,we sorted MDSC from spleen of WT and Siglecg/-tumor-bearing mice and co-cultured them with anti-CD3/CD28 activated CD4+/CD8+T cells at a ratio of 1:2.Two days later,the proliferation of CD4+T/CD8+T cells was detected by Ki67 staining.The expression of FasL on CD8+T cells was detected by flow cytometry.Splenic MDSC from Siglecg-/-tumor-bearing mice did not affect the proliferation of CD4+T/CD8+T cells or the expression of FasL,Perforin and GzmB on CD8+T cells.However,its ability to inhibit the secretion of IFN-y by CD8+T cells was enhanced.After adding TGF-β or IL-10 neutralizing antibody,WT and Siglecg-/-MDSC showed no significant difference in inhibiting CD8+T cell secretion of IFN-y,suggesting that Siglecg-/-MDSC inhibited CD8+T cell secretion of IFN-γ through TGF-β or IL-10.These results suggest that Siglec-G deficiency alters the function of MDSC in the spleen of tumor-bearing mice.Will Siglec-G deficiency lead to similar changes in the function of MDSC induced in vitro?We took bone marrow cells from WT and Siglecg-/-mice and induced MDSC in vitro with GM-CSF.After 72 hours,MDSC were sorted and examined for their cytokines secretion and their effect on IFN-γ secretion by activated CD8+T cells.Compared with WT MDSC,Siglecg-/-MDSC induced in vitro secreted more TGF-β and IL-10,and their ability to inhibit IFN-y secretion by CD8+T cells was enhanced.After adding the neutralizing antibody of TGF-β or IL-10,Siglecg-/-MDSC lost its ability to inhibit IFN-y secretion by CD8+T cells.These results suggest that Siglec-G deficiency can also enhance the immunosuppressive function of MDSC induced in vitro.To explore the effect of Siglecg-/-MDSC on tumor progression,we sorted MDSC from the spleen of WT and Siglecg-/-tumor-bearing mice and adoptive transfused them back into B16F 10-bearing mice through the tail vein.The tumor growth of the mice was observed.Compared with the adoptive return of WT MDSC,the adoptive return of Siglecg-/-MDSC showed faster tumor growth,heavier tumor weight and larger tumor size.To explore whether Siglec-G deficiency also affects the proportion and function of MDSC in tumor tissues,the proportion and quantity of MDSC and its subsets in tumor tissues were detected by flow cytometry and immunofluorescence.The results showed that compared with WT mice,the proportion and number of MDSC in the tumor tissues of Siglecg-/-mice were increased,and the proportion of PMN-MDSC was increased,while M-MDSC was unchanged.Moreover,the expression of membrane TGF-β and CXCR2 was increased.The above results suggest that Siglec-G deficiency may promote the chemotaxis of spleen MDSC to tumor tissues,and further experiments are needed to verify it.We initially explored the mechanism by which Siglec-G deficiency regulates MDSC function.We first examined the expression of Siglec-G in MDSC.The expression of SiglecG in CD11b+ Gr-1+ cells in liver,spleen,bone marrow and peripheral blood of normal mice,B16F10 tumor-bearing mice and MC38 tumor-bearing mice was detected by flow cytometry.The results showed that Siglec-G was expressed in CD11b+Gr-1+ cells in the above tissues.These results suggest that Siglec-G deficiency may directly affect the related functions of MDSC.WT and Siglecg-/-mice were subcutaneously injected with B16F10 melanoma cell line.Single cell suspensions were prepared from spleens of the mice 17 days after injection.KEGG database analysis showed that Siglecg-/-MDSC related MAPK pathway was activated.We sorted the spleen MDSC from WT and Siglecg-/-B16F10 tumor-bearing mice,extracted total proteins,and detected the activation of related pathways by Western Blot.The results showed that p-ERK was highly expressed in Siglecg-/-MDSC compared with WT MDSC.After MDSC were treated with ERK inhibitor SCH772984 for 6 h,the secretion of TGF-β was detected by intracellular staining and flow cytometry.The results showed that inhibition of ERK decreased the secretion of TGF-β by Siglecg-/-MDSC.These results suggest that SiglecG deficiency increases the phosphorylation of ERK in MDSC,which promotes the expression of TGF-β.In summary,this study found that Siglec-G deficiency reduces MDSC in spleen,upregulates its immunosuppressive function,and plays a role in promoting tumor.