In vitro effects of G-CSF, GM-CSF, and M-CSF treatments on expansion and suppressive function of murine bone marrow-derived myeloid-derived suppressor cells
Introduction: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that can suppress the adaptive and innate immune responses and upregulate other downregulatory elements of the immune system. MDSCs may help to suppress graft-versus-host disease (GVHD) in allogeneic transplant recipients. GVHD is a complication following allogeneic stem cell transplantation in which graft T cells launch an immunological response against recipient tissues. Objective: We wished to identify a growth factor (GF) that promoted and expanded a suppressive MDSC population. Methods: To test the ability of myeloid GFs to maintain, expand and improve suppressive function of bone marrow (BM)-derived MDSCs we cultured murine BM with G-CSF, GM- CSF or M-CSF. Cells were stained with fluorescent antibodies to surface antigens that are expressed by MDSCs and measured by flow cytometry. Suppressive function of in vitro GF-treated BM was measured by inhibition of third party CFSE-labeled splenocyte proliferation. The effects of blocking MDSC suppressive function by adding inhibitors were evaluated. Comparisons were made between GF treatment groups using one-way ANOVA tests and two-way ANOVA tests with Tukey’s range tests. Results: 4 days of culture of BM with 10 ng/ml GM-CSF, 20 ng/ml G-CSF or 20 ng/ml M-CSF generated high numbers of live MDSC, of which GM-CSF stimulation generated the highest number. MDSCs induced by different GF can suppress the proliferation of CD4+ T lymphocytes in dose-dependent manner, of which M-CSF- treated murine BM showed greatest suppressive function (suppression rates for 1:1 co-culture are G-CSF 12%[SD=4], GM-CSF 72.9%[0.5], M-CSF 83%, no GF 24%; adjusted P values < 0.0001 for all comparisons except P=0.0104 G-CSF vs. no GF and P=0.02 for GM-CSF vs. M- CSF using 2-way ANOVA). M-CSF-treated BM promoted the greatest regulatory T cell expansion in co-culture. Addition of an iNOS inhibitor, not the inhibitors to M-CSF-treated BM and splenocyte co-culture appeared to reverse the suppressive function of this cell population. Conclusion: M-CSF-induced MDSCs, as well as GM-CSF-induced MDSCs, had a strong suppressive effect on the proliferation of murine splenocytes, and may through an iNOS pathway. The use of M-CSF in the post-transplant setting may be a new tool for the treatment of GVHD.
Myeloid-derived suppressor cells (MDSCs), graft-versus-host disease (GVHD)