Myeloid-Derived Suppressor Cells Are Expanded in Patients with AML and Are Dependent on MUC1 Expression

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive milieu of the tumor microenvironment and play an important role in promoting immune tolerance and disease growth. They are comprised of granulocytic and monocytic compartments defined by a unique immunophenotypic signature. Importantly, the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. In the present study, we examined the interaction of MDSCs with AML cells, a setting in which the presence and function of MDSC has not been well described. Methods and Results: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with active AML and granulocytic (CD33+/CD11b+/HLADR-/CD15+) and monocytic (CD33+/CD11b+/HLADR-/CD15-) MDSCs were quantified by multichannel flow cytometry. AML patients had a significantly higher mean granulocytic MDSC population of 17.2% (n=3) compared to healthy controls 1.9%, (n=10) p=0.0083 and a mean monocytic MDSC population of 6.5% (n=3), which was similar to healthy controls (monocytic MDSCs 4.1%, n=10). MDSCs isolated from an AML patient exhibited immunosuppressive effects as measured by the suppression of dendritic cell mediated stimulation of T cells. The addition of AML derived MDSCs resulted in a 40% reduction in CD4+T cell production of IFNϒ and an 11 fold increase IL-10 secretion by CD4 and CD8 T cells following coculture with allogenic DC stimulation. The ability of AML blasts to directly induce the expansion of MDSC was assessed in vitro. Healthy donor PBMCs were co-cultured for 6 days with or without the AML cell lines MOLM-14 and THP-1 at a ratio of 100:1. MDSCs were quantified after 6 days. Coculture with MOLM-14 and THP-1 induced a 2.35 and 8.2 fold increase in MDSCs respectively (n=4). MUC1 is a critical oncogene expressed on leukemic blasts and leukemia initiating cells and plays an important role in the tumor microenvironment promoting tumor growth and immune escape. In the present study, we demonstrated that silencing of MUC1 via shRNA significantly diminishes AML recruitment and expansion of MDSCs in vitro. MOLM-14 cells underwent lentiviral transfection to silence MUC1-C expression which was confirmed by Western Blot. MOLM-14 wild type, MUC1 silenced, and control vector treated cells were co-cultured with healthy PBMCs for 6 days in a ratio of 100:1. Of note, MUC1-C silenced MOLM-14 and THP-1 cells exhibited decreased capacity to expand MDSCs upon co-culture with healthy donor PBMCs, as compared to the control vector (2.4 fold higher expansion of MDSCs with control vector MOLM-14 compared to MUC1-C silenced MOLM-14, n=4, 1.92 fold higher expansion of MDSCs with control vector THP-1 compared to MUC-1C silenced THP-1, n=4). In an in vivo model, NSG mice were irradiated and inoculated with THP-1 control and THP-1 MUC1 silenced cells. Following establishment of disease, mice were sacrificed and spleens were FACS analysed for MDSC quantification. Mice inoculated with THP-1 MUC1 silenced cells had mean MDSCs of 7.5%, compared to 16.25% in mice innoculated with THP-1 Wildtype cells (n=4). In conclusion, the data demonstrates that MDSCs are increased in the circulation of patients with AML, and that leukemic blasts directly induce the expansion of MDSCs. MUC1 expression on AML blasts contributes to the immunosuppressive milieu, and notably, silencing of MUC1 in AML cells blunts their capacity to induce the expansion of MDSCs. Incorporating strategies to inhibit the expansion of MDSC in AML, and reverse their immunosuppressive phenotype has the potential to improve response to therapy in AML. Disclosures No relevant conflicts of interest to declare.