Figure 6

Morphology, phenotype, and function of CD11b⁺ and CD33⁺ MDSC subsets. A, Morphology of human CD33⁺ and CD11b⁺ MDSC subsets isolated after tumor cell line co-culture and normal myeloid counterparts from medium only cultures (Wright-Giemsa staining, x400, original magnification). CD33⁺ MDSC appear slightly more differentiated than CD11b⁺ MDSC after induction. Images are representative of data from more than five donors and three independent experiments using SCCL-MT1 or USC-HN2 for CD33⁺ MDSC induction and MCF7 or NCI-H60 for CD11b⁺ MDSC induction. B, Human CD33⁺ and CD11b⁺ MDSC are distinct subsets with a common HLA-DR^low Lineage^- phenotype. Phenotype of HNSCC cell line-induced CD33⁺ and breast cancer cell line-induced CD11b⁺ MDSC compared with medium only, non-suppressive CD33⁺ and CD11b⁺ cells as measured by flow cytometry. Mean percent positive cells (n ≥ 2) + SD shown, data from three unique donors. Differences in percent positive cells analyzed by ANOVA then Bonferroni's multiple comparison test for selected pairs (* indicates statistically significant difference in mean percent positive between MDSC and medium control for each subset, p <0.05). C, Comparison of ARG-1, iNOS, and NOX2-component NCF1 gene expression in CD33⁺ and CD11b⁺ human MDSC revealed similar levels of expression between these subsets. Mean fold change shown relative to medium only controls (n = 3 unique donors for MDSC from co-cultures with each of three inducing tumor models) + SEM. No statistically significant difference between means as determined by Student's t test for each gene. D, Elevated arginase activity (left) and reactive oxygen species (ROS) production (right) by tumor cell line-induced CD33⁺ and CD11b⁺ MDSC. Arginase activity of CD11b⁺ and CD33⁺ MDSC subsets as measured by arginine degradation to urea and compared with normal myeloid cells. Mean shown + SEM; data from four unique donors and two inducing cancer cell lines for each subset. * indicates statistical significance (p <0.05); NS = not significant. ROS production by CD11b⁺ and CD33⁺ MDSC subsets as measured by DCFDA and compared with normal myeloid cells. Mean fluorescence intensity shown + SEM for 20,000 events collected; data from three unique donors and two inducing cancer cell lines for each subset. No significant difference by ANOVA.