Abstract 4741: Monocytic myeloid derived suppressor cells (M-MDSC) from spleen are multipotent while tumor M-MDSC have limited plasticity

Abstract

Abstract The tumor (Tu) microenvironment contains monocytic myeloid-derived suppressor cells (M-MDSC, CD11b+Ly6ChiLy6Glo) that suppress cytotoxic T cell-mediated immune surveillance. M-MDSC-like cells are also found in the spleen of Tu-bearing mice (Sp) and are often used as surrogates for Tu MDSC. We conducted a series of experiments to test if these two cell types are functionally similar. Sp- and Tu M-MDSC were isolated and used in a short-term T cell suppression assay (16 h) but only Tu M-MDSC could suppress T cell proliferation. Next, we examined the differences between the Sp and Tu by examining cells with M-MDSC or granulocytic-MDSC (G-MDSC; CD11b+Ly6CmedLy6Ghi) markers using Affymetrix Mouse Gene 1.0 ST microarrays. More than 5600 differentially expressed transcripts (1.5 fold, 5% FDR) were seen between the Sp and Tu within each MDSC subtype; 1451 of these transcripts were unique for the M-MDSC type. While others have shown that Sp M-MDSC can differentiate toward other end-stage myeloid cells, it is not clear whether this plasticity extends to Tu M-MDSC. Thus, we tested the ability of M-MDSC cells from Tu or Sp to differentiate into osteoclasts (Oc; RANKL and M-CSF) and immune cells found in the Tu microenvironment: G-MDSC (GM-CSF), dendritic cells (DC; GM-CSF and IL-4), or macrophages (Mϕ; M-CSF). Upon treatment, Sp M-MDSC acquired the markers of Oc (RANK), G-MDSC (loss of Ly6C, gain of Ly6G), DC (CD11c), and Mϕ (F4/80). In contrast, Tu M-MDSC could differentiate into Mϕ, but not into Oc, DC or G-MDSC. When Tu M-MDSC were cultured with both tumor explant supernatant (TES, to mimic the Tu microenvironment) and differentiating agents their capacity to differentiate did not significantly change. However, TES-treated Sp M-MDSC became less responsive to signals for conversion to G-MDSC and DC and more responsive to the Mϕ-inducing treatment. Our data suggest that Sp M-MDSC are immature, multipotent cells and addition of TES causes them to mature towards a less plastic Tu M-MDSC. Finally, we tested whether the functional differences among naïve bone marrow, Sp, and Tu cells were detectable from morphological (FSC-A, SSC-A) or cell marker data (CD11b, Ly6C, Ly6G) from flow cytometry. Singlet myeloid cells were selected via automated gating and continuous phenotype data was subjected to unsupervised K- means clustering to create unique templates of cell populations for each tissue site (FlowMatch). An n-1 cross-validation approach tested the accuracy of the classification and showed that the 3 templates correctly classified each sample to its tissue of origin. Thus, multidimensional flow cytometry data reflects the functional differences between CD11b+Ly6ChiLy6Glo cells from different tissues. The major finding of these experiments is that Tu M-MDSC are a more mature phenotype that are morphologically and functionally distinct from the M-MDSC precursors found in Sp. Citation Format: Ryan D. Calvert, James C. Fleet, Ye Chen, Alex Pothen, Bartek Rajwa, Pierrick G. Fournier, Patricia Juarez, Theresa A. Guise, Timothy L. Ratliff, Ben D. Elzey. Monocytic myeloid derived suppressor cells (M-MDSC) from spleen are multipotent while tumor M-MDSC have limited plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4741.