FIGURE 2 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

PMN-MDSCs develop from monocytes and immature progenitors. A, Representative flow cytometry histogram of CFSE dilution in total monocytes (CD11b+MHCIInegLy6C+) and total PMN (CD11b+MHCIInegLy6G+) on days 1 and 4 in culture ± 1.5 µmol/L DAB. Day 4 panels show percent (%) of CFSEhigh cells. The purple shaded area shows PMN cells that remain quiescent and do not proliferate beyond the initial cycle on day 1. The green shaded area shows PMN cells that are highly proliferative. B, Representative flow cytometry contour plot of Ly6C and Ly6G expression in total PMNs gated as CD11b+MHCIInegLy6G+ on day 4 after differentiation ± 1.5 µmol/L DAB. Quiescent (purple) and proliferative (green) PMN cells described in A are overlaid for each condition. C, log2 fold change (log2FC) of cell counts of total monocytes (CD11b+MHCIInegLy6C+) and total PMNs (CD11b+MHCIInegLy6G+) on day 4 normalized to the seeding counts of each lineage group at basal timepoint. MDSC were generated in presence of ± 1.5 µmol/L DAB. P values were determined by two-way ANOVA with Šidák correction post hoc-test. Significance considered P D, log2FC in proliferation measured by incorporation of DNA intercalating fluorescent dye on day 4, normalized to the fluorescence signal of cells at baseline. Sorted monocytes (CD3negCD19negCD11b+MHCIInegLy6C+LyG6neg), PMN (CD3negCD19negCD11b+MHCIInegLy6CmedLyG6+) or progenitors (CD5negCD11bnegCD19neg B220negGr-1negTER119neg) were differentiated to MDSC ± 1.5 µmol/L DAB. Cells were sorted from fresh bone marrow and plated at the same cell density (2 × 104 cells/well). Bars show mean ± SD (n = 4). P values were determined by two-way ANOVA with Šidák correction post-test. Significance considered P E, Representative flow cytometry contour plot of Ly6C and Ly6G expression in CD11b+MHCIIneg cells differentiated for 4 days. Initial cell source was either sorted bone marrow monocytes (CD3negCD19negCD11b+MHCIInegLy6C+LyG6neg), bone marrow PMNs (CD3negCD19negCD11b+MHCIInegLy6CmedLyG6+) or bone marrow progenitors (CD5negCD11bnegCD19neg B220negGr-1negTER119neg). Gates show mean frequency. F, Frequency of total CD11b+ cells during sorted progenitor differentiation, assessed by flow cytometry for each day of cell differentiation. Line represents mean ± SD (n = 4). G, Representative contour plot of Ly6C and Ly6G expression in CD11b+MHCIIneg cells each day during sorted progenitor differentiation. Gates show mean frequency. Experiment repeated three times with similar results. H, Frequency of the granulocytic population CD11b+MHCIInegLy6C+Ly6G+ during progenitor differentiation, determined by flow cytometry for each day of cell differentiation. Bars indicate mean ± SD (n = 4). P values were determined by two-way ANOVA with Šidák correction post-test. Significance considered P