NKG2D Ligand RAE1? Induces Myeloid-Derived Suppressor Cells Generation And The Underlying Mechanisms In Mice

Myeloid-derived suppressor cells(MDSCs)represent a heterogeneous population of myeloid progenitor cells with immune suppressive function and has emerged as one of the central regulators of immune responses in cancer.The phenotype of MDSCs in mice is CD11b+Gr-1+.Tumor cells can secret some cytokines,such as IL-6,TGF-? and IL-33,to induce MDSCs generation.However,little is known about whether tumor surface molecules can induce MDSCs generation or not.Ligands for NKG2D,such as RAE1?(RAE1?-RAE1?),are expressed on tumor cells.Ligand activation of NKG2D plays key roles in antitumor immunity through activating CD8+ T cells and NK cells.However,tumor cells are able to escape from the NKG2D-mediated killing by down-regulating NKG2D expression on cytolytic cells.Up to now,very limited information is available regarding NKG2D ligands inducing MDSCs generation.Therefore,in this study,we investigate whether NKG2D ligand RAE1? can induce MDSCs generation in vitro and in vivo and what are the underlying mechanisms.In previous study,a BaF3-RAE1? cell that expressed RAE1? and a BaF3-mock control cell with empty plasmid were structured.To explore the role of RAE1? on the generation of CD11b+Gr-1+ cells,BaF3-mock or BaF3-RAEle cells were injected into mice,and the absolute number and the percentage of CD11b+Gr-1+ cells were detected.The results showed that the percent and absolute number of CD11b+Gr-1+ cells were significantly increased in spleen and blood of BaF3-RAE1? tumor-bearing mice.NKG2D blockade using neutralizing anti-NKG2D antibody in BaF3-RAE1? tumor-bearing mice significantly reduced the CD11b+Gr-1+ cell levels,indicating that RAE1? mediates these cells generation via NKG2D in vivo.To explore whether RAE1? induces the generation of CD11b+Gr-1+ cells in vitro,bone marrow cells were cultured with CFSE-labelled BaF3-mock or BaF3-RAE1? for 5 days and then the percentage and the absolute number of CD11b+Gr-1+ cells were detected in CFSE negative cells.We found that RAE1? induced the CD11b+Gr-1+ cell generation and blocking of NKG2D using neutralizing anti-NKG2D antibodies led to decrease in CD 11 b+Gr1+ cell levels.Thus,we conclude thatRAE1? mediates CD11b+Gr-1+ cells generation via NKG2D in vivo and in vitro.To explore whether CD11b+Gr-1+ cells induced by BaF3-RAE1? are functional distinct from CDllb+Gr-1+ cells induced by BaF3-mock,mice were injected with BaF3-mock or BaF3-RAElscells,and splenic CD11b+ cells were sorted by magnetic beads.We found that there was a significant up-regulation in the arginase activity and IL-10 secretion of CD11b+ cells induced by RAE1?,while there was no significant change in the expressions of PGE2 and NO.BaF3-RAE1?-derived CD11b+ cells possessed a more intense inhibitory effect on the proliferation of CD8+ T cells,and nor-NOHA(arginase inhibitor)treatment or IL-10 blockade could significantly reverse the suppression of the activated CD8+ T-cell proliferation.Taken together,these results demonstrated that RAE1? induces splenic MDSCs with enhanced suppressive functions and these functions are arginase and IL-10-dependent.Furthermore,we also determined several related cytokines in the sera of BaF3-RAE1? and BaF3-mock tumor-bearing mice.We found that IL-4 was increased in the sera of BaF3-RAE1? tumor-bearing mice compared with BaF3-mock tumor-bearing mice,while there was no significant change in the IL-6 and IFN-y.To explore whether MDSCs induced by RAE1? possesses enhanced pro-tumor effect,mice were subcutaneously transplanted with CT-26 tumor cells and then adoptive transfer of BaF3-mock-derived MDSCs or BaF3-RAE1?-derived MDSCs.We found that the adoptive transfer of BaF3-RAE1?-derived MDSCs significantly enhanced the growth of subcutaneous tumors and diminished the survival rate of the mice.A delay in tumor growth was observed as long as nor-NOHA or anti-IL-10 was administered,suggesting that BaF3-RAE1? MDSCs promote tumor growth in arginase-and IL-10-dependent manners.These results suggesting that RAE1?-induced MDSCs can promote the tumor growth via IL-10 and arginase.To explore whether RAE1? is directly enhancing the suppressive function of MDSCs in vitro,spleen MDSCs derived from CT-26 tumor-bearing mice were co-cultured with BaF3-RAE1? or BaF3-mock,and then the immunosuppressive functions of MDSCs were detected.We found that MDSCs co-cultured with BaF3-RAE1? produced higher levels of IL-10 and IL-4.Blocking of RAE1?-NKG2D interaction impaired IL-4 and IL-10 production.Furthermore,MDSCs co-cultured with BaF3-RAE1? significantly inhibited CD8+ T-cell proliferation via IL-10.In conclusion,we show that NKG2D ligand RAE1? facilitates generation and activation of immunosuppressive MDSCs in vitro and in vivo,thereby providing a new mechanism by which NKG2D ligands inhibit immune response and lead to tumor progression.