Fc?R?b Deficiency Inhibits The Negative Immunomodulatory Function Of MDSC Subsets

Myeloid-derived suppressor cell(MDSC)is a group of heterogeneous cells with strong immunosuppressive function,which is negatively correlated with the prognosis of various tumors and reduces the effect of tumor immunotherapy.The Fc receptor(Fc?R)of immunoglobulin IgG consists of two families with opposite functions of activation and inhibition.There are many types of activating Fc?R,while there is only one type of inhibitory FcyR in humans and mice,namely Fc?RIIb,which is widely expressed on the surface of B cells and myeloid cells,and mediates its immunoregulatory functions.However,whether myeloid-derived MDSCs express Fc?R?b?If expressed,can Fc?RIIb regulate the differentiation and function of MDSC?Our previous studies showed that MDSC in tumor-bearing mice expresses FcyRIIb,and FcyRIIb deficiency has a series of effects on the differentiation and function of MDSCs.Compared with wild-type(WT)tumor-bearing mice,FcyRIIb defective(FcyRIIb-/-)tumor-bearing mice have more CD11b+Gr-1+MDSCs in spleen.Fc?RIIb-/-splenic MDSCs expressed lower quantities of ARG-1(arginase-1)and IL-10,but higher amounts of iNOS(inducible nitric oxide synthase)and TNF-?(tumor necrosis factor ?)than WT MDSCs.Functionally,the abilities of Fc?RIIb-/-splenic MDSCs for inhibiting the proliferation of activated CD4+T cells and inducing Tregs significantly decreased compared with those of MDSCs from WT tumor-bearing mice.These results suggesting that Fc?RIIb deficiency inhibits the immunosuppressive function of MDSC.It is known that MDSC is divided into two subsets:granulocyte-type MDSC(PMN-MDSC with CD11b+ly6G+ly6Clow phenotype)and monocyte-type MDSC(M-MDSC with CD11b+ly6G-ly6Chi phenotype),PMN-MDSC mainly inhibits the activation of T cells by up-regulating the expression of reactive oxygen species(ROS),while M-MDSC inhibits the function of T cells by producing ARG-1.However,which subset of MDSCs regulated by FcyRIIb and what the function of Fc?RIIb-/-MDSCs subsets in tumor progression have not been elucidated.Therefore,in this study,we investigated the regulatory effect of FcyRIIb deficiency on the function of MDSC subsets.In order to investigate the effects of FcyRIIb on the amplification and function of MDSC subsets,we used WT and Fc?RIIb-/-mice to prepare tumor-bearing models of 3LL lung cancer cell line and B16F10 melanoma cell line,respectively.The ratio and absolute number of MDSC subpopulations in the spleen of WT and Fc?RIIb-/-tumor-bearing mice were detected by fluorescent particle counting method.The results showed that the proportion and number of Fc?RIIb-/-PMN-MDSC in the spleen of tumor-bearing mice were significantly increased,while the number of FcyRIIb-/-M-MDSC was significantly increased compared with those of WT tumor-bearing mice but the proportion was not significantly different.In order to investigate whether the FcyRIIb deficiency affects the enzymatic activity and cytokine expression of MDSC subsets in the spleen,we sorted PMN-MDSC and M-MDSC in the spleen of WT and Fc?R?b-/-tumor-bearing mice by flow cytometry and detected the ARG-1,TGF-?,IL-10 and TNF-? expression levels.The results showed that Fc?R?b-/-PMN-MDSC had lower quantities of ARG-1,IL-10 and TGF-? expression than WT PMN-MDSC,while TNF-? expression had no obvious difference.Moreover,Fc?R?b-/-M-MDSC had lower expression of ARG-1 and IL-10 and more expression of TNF-? than WT M-MDSC,while TGF-? expression had no obvious difference.In order to investigate whether the FcyRIIb deficiency affects the function of MDSC subsets in the spleen,WT and Fc?R?b-/-PMN-MDSC or M-MDSC were sorted and co-cultured with activated CD4+T cells or CFSE-labeled activated CD4+T cells,and then the CD4+T cell proliferation and Treg population were examined by flow cytometry.The results showed that the ability of PMN-MDSC and M-MDSC from Fc?R?b-/-tumor-bearing mice for inhibiting the proliferation of activated CD4+T cells significantly decreased compared with that of PMN-MDSC and M-MDSC from WT tumor-bearing mice.Furthermore,decreased Treg cell induction was observed when comparing Fc?R?b-/-to WT splenic M-MDSC.The ability of PMN-MDSC to induce Treg was not significantly changed.The above results suggest that the deficiency of FcyRIIb leads to weakened immunosuppressive function of the MDSC subsets.In order to investigate the role of Fc?R?b-/-MDSC subsets in tumor progression,mice were subcutaneously transplanted with B16F10 tumor cells and then adoptive transfer of WT MDSC subsets or Fc?R?b-/-MDSC subsets.The results showed that upon adoptive transfer,Fc?R?b-/-MDSC subsets significantly retarded the growth of subcutaneous tumors and prolonged survival rate compared with WT MDSC subsets in B16F10 tumor bearing mice.The above results suggest that FcyRIIb deficiency leads to the obvious inhibition of the tumor-promoting effects of PMN-MDSC and M-MDSC.In order to investigate the effect of FcyRIIb deficiency on the proportion and function of MDSCs induced in vitro,we added GM-CSF in WT and Fc?R?b-/-bone marrow cells to induce MDSC production.After 24 hours,the MDSC,PMN-MDSC and M-MDSC populations were detected in the bone marrow cells by flow cytometry.The results showed that the proportion of MDSC,PMN-MDSC,and M-MDSC in bone marrow cells from Fc?R?b-/-mice was significantly increased compared with that of WT mice.In addition,we sorted GM-CSF-induced MDSCs and measured the expression levels of related cytokines and enzymes.The results showed that Fc?R?b-/-MDSC had decreased expression of ARG-1 and TNF-? than WT MDSC.The expression of TGF-? was not significantly changed.The above results suggest that FcyRIIb deficiency also affects the proportion and function of MDSCs induced in vitro.In conclusion,we show that FcyRIIb deficiency can inhibit the immunosuppressive function of PMN-MDSC and M-MDSC,and the results obtained from this project might be helpful to enrich our understanding of MDSC subsets plasticity and open novel avenues for therapeutic strategies.