Objective·To explore the regulatory mechanism of folate cycle metabolism in the immunosuppressive effect of myeloid derived suppressor cells(MDSCs).Methods·Bone marrow cells were isolated from C57BL/6 mice and cultured in RPMI 1640 medium supplemented with GM-CSF,G-CSF,and IL-6 to induce MDSCs in vitro.PD-L1 expression level and ROS production level of induced MDSCs were detected by flow cytometry.CD8+T cells were enriched from the spleen by MACS with anti-CD8a-conjugated microbeads,labeled with Celltrace violet,and then co-cultured with MDSCs.After 72 h,proliferation was assessed by flow cytometry.Folate cycle-related metabolic enzymes in MDSCs were detected by real-time quantitative PCR.MDSCs were treated with folate cycle metabolic enzyme MTHFD2 inhibitor DS18561882(DS18)and folic acid antagonist Pemetrexed.ROS and mitoROS production in MDSCs were assessed by flow cytometry.CD8+T cells were enriched from the spleen by MACS with anti-CD8a-conjugated microbeads,labeled with Celltrace violet,and then co-cultured with Pemetrexed or DS18-treated MDSCs.After 72 h,proliferation was assessed by flow cytometry.Transcript levels of folate cycle-related metabolic enzymes in pemetrexed or DS18-treated MDSCs were detected by RNAseq.A subcutaneous tumor mouse model of colon cancer was established.From the tenth day post-implantation,tumor-bearing mice were intraperitoneally injected with Pemetrexed(200 mg/kg)and tumor size was recorded for tumor growth curve.On the fourteenth day,mice were sacrificed,and tumors were harvested.MC38 tumor-bearing mice were treated with isotype antibody,anti-CD8 monoclonal antibody(1 mg/kg,deplete CD8+T cells),Pemetrexed(200 mg/kg),and combination of Pemetrexed with anti-CD8 antibody.MC38 tumor-bearing mice were treated with isotype antibody,anti-Gr1 monoclonal antibody(1.25 mg/kg,clearing MDSCs),combination of Pemetrexed with anti-Gr1 antibody.On the tenth day after implantation,tumor-bearing mice were treated with Pemetrexed(50 mg/kg),anti-PD-1 monoclonal antibody(250 μg/kg),Pemetrexed,and combination of Pemetrexed with anti-PD-1 antibody.Results·Flow cytometry data showed that PD-L1 level and ROS production were increased in induced MDSCs,and CD8+T cell proliferation was also suppressed significantly.qPCR data revealed the expression of folate cycle-related metabolic enzymes MTHFD2 and others was increased in MDSCs.The accumulation of MDSCs was affected by DS18 or Pemetrexed,ROS production in MDSCs was reduced,and the immunosuppression of CD8+T cells was relieved.RNA-seq results showed that genes related to MDSCs differentiation,such as S100 calc-binding protein A8,and genes related to MDSCs inhibition,such as cytochrome b-245β chain,which is related to ROS production,were also down-regulated after treatment with two folic acid cycling inhibitors.Tumor growth was suppressed by Pemetrexed.Tumor progression was promoted by combination of Pemetrexed with anti-CD8 antibody,compared with Pemetrexed monotherapy.However,tumor growth delay was inhibited by combination of Pemetrexed and anti-CD8,compared with anti-CD8 monotherapy.Tumor growth delay was caused by MDSCs depletion.But tumor growth was promoted by combination of pemetrexed and anti-Gr1,compared with pemetrexed monotherapy.Tumor growth was restricted by combination of pemetrexed and anti-PD-1 antibody,compared with anti-PD-1 monotherapy.Conclusion·Pemetrexed relies on CD8+T cells for anti-tumor effects and further retards tumor growth by reprogramming MDSCs to an anti-tumor phenotype.Modulating MDSCs by targeting folate cycle could impair their immunosuppressive ability and enhance the efficacy of immune checkpoint blockade in cancer treatment.