Modified method for differentiation of myeloid-derived suppressor cells in vitro enhances immunosuppressive ability via glutathione metabolism

Haoyang Zhou; Zhiqi Xie; Naosuke Morikawa; Fuminori Sakurai; Hiroyuki Mizuguchi; Daisuke Okuzaki; Naoki Okada; Masashi Tachibana

doi:10.1016/j.bbrep.2022.101416

Modified method for differentiation of myeloid-derived suppressor cells in vitro enhances immunosuppressive ability via glutathione metabolism

Biochem Biophys Rep. 2022 Dec 28:33:101416. doi: 10.1016/j.bbrep.2022.101416. eCollection 2023 Mar.

Authors

Haoyang Zhou¹, Zhiqi Xie^{1

2}, Naosuke Morikawa³, Fuminori Sakurai³, Hiroyuki Mizuguchi^{3

4

5

6

7}, Daisuke Okuzaki^{5

7

8

9}, Naoki Okada^{1

10}, Masashi Tachibana^{1

3

6

10}

Affiliations

¹ Project for Vaccine and Immune Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
² Department of Pharmacology, School of Medicine, Zhejiang University City College, Zhejiang, 310015, China.
³ Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
⁴ Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, 567-0085, Japan.
⁵ Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, 565-0871, Osaka, Japan.
⁶ Global Center for Medical Engineering and Informatics, Osaka University, Osaka, 565-0871, Japan.
⁷ Center for Infectious Disease Education and Research, Osaka University, Osaka, 65-0871, Japan.
⁸ Single Cell Genomics, Human Immunology, WPI Immunology Frontier Research Center, Osaka University, Osaka, 65-0871, Japan.
⁹ Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan.
¹⁰ Laboratory of Vaccine and Immune Regulation (BIKEN), Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.

Abstract

Myeloid-derived suppressor cells (MDSCs), which accumulate in tumor bearers, are known to suppress anti-tumor immunity and thus promote tumor progression. MDSCs are considered a major cause of resistance against immune checkpoint inhibitors in patients with cancer. Therefore, MDSCs are potential targets in cancer immunotherapy. In this study, we modified an in vitro method of MDSC differentiation. Upon stimulating bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor in vitro, we obtained both lymphocyte antigen 6G positive (Ly-6G⁺) and negative (Ly-6G^-) MDSCs (collectively, hereafter referred to as conventional MDSCs), which were non-immunosuppressive and immunosuppressive, respectively. We then found that MDSCs differentiated from Ly-6G^- BM (hereafter called 6G^- BM-MDSC) suppressed T-cell proliferation more strongly than conventional MDSCs, whereas the cells differentiated from Ly-6G⁺ BM (hereafter called 6G⁺ BM-MDSC) were non-immunosuppressive. In line with this, conventional MDSCs or 6G^- BM-MDSC, but not 6G⁺ BM-MDSC, promoted tumor progression in tumor-bearing mice. Moreover, we identified that activated glutathione metabolism was responsible for the enhanced immunosuppressive ability of 6G^- BM-MDSC. Finally, we showed that Ly-6G⁺ cells in 6G^- BM-MDSC, which exhibited weak immunosuppression, expressed higher levels of Cybb mRNA, an immunosuppressive gene of MDSCs, than 6G⁺ BM-MDSC. Together, these data suggest that the depletion of Ly-6G⁺ cells from the BM cells leads to differentiation of immunosuppressive Ly-6G⁺ MDSCs. In summary, we propose a better method for MDSC differentiation in vitro. Moreover, our findings contribute to the understanding of MDSC subpopulations and provide a basis for further research on MDSCs.

Keywords: Ab, antibody; BM, bone marrow; BM-MDSC; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; Cybb, Cytochrome b-245 beta polypeptide; FBS, fetal bovine serum; GM-CSF, granulocyte-macrophage colony-stimulating factor; Glutathione metabolism; ICI, immune checkpoint inhibitor; Immunosuppression; Ly-6G; Ly-6G, lymphocyte antigen 6G; M-MDSCs, monocytic MDSCs; MDSCs, myeloid-derived suppressor cells; Myeloid-derived suppressor cell; PBS, phosphate-buffered saline; PD-1, programmed cell death 1; PD-L1, programmed cell death 1 ligand 1; PMN-MDSCs, polymorphonuclear MDSCs; ROS, reactive oxygen species; Rb1, retinoblastoma 1; Tumor progression; iNOS, inducible nitric oxide synthase.