American Society of Hematology

Figure 2

$Figure 2. PGE2 redirects DC differentiation and induces CD14+CD33+CD34+ cells with the phenotype and function of monocytic MDSCs isolated from cancer patients. (A) Phenotype of PGE2-induced CD1a−CD14+CD80−CD83− MDSCs expressing inhibitory molecules ILT2, ILT3, ILT4, PDL-1, but not PDL-2. PGE2-induced MDSCs express E-prostanoid receptors (labeled with α-EP1–, α-EP3–, sec.Alexa488, α-EP2–, and α-EP4–PE). (B) Expression of immunosuppressive factors IL-10, IDO1, IL-4Rα, and COX2 in PGE2-induced MDSCs (see supplemental Figure 1A for corresponding protein levels of IDO and IL-10). (C) Immunosuppressive effects of PGE2-induced MDSCs on allogeneic naive CFSE-labeled CD8+ T cells primed by CD3/CD28 and stained for granzyme B. Left panel: Percentages indicate the fraction of proliferating granzyme B+ (marker of CTL status) CD8+ cells. Right panel: Percentage of proliferating CD8+ T cells in the presence of PGE2-induced MDSCs (PGE2-d0) and PGE2-conditioned DCs (PGE2-d6). (D-F) MDSC phenotype and function of CD11b+ cells isolated from cancer ascites. (D) Characterization of cells from cancer ascites either before (left panel) or after (right panel box) isolation of CD11b+ cells. Note the high percentage of CD11b+ cells (8.9%-50.0%, mean 24.2%, n = 7) within the cancer-infiltrating primary cell population. (E) mRNA levels of IL-10, IDO1, IL-4Rα, and COX2 in CD11b+ cells isolated from cancer (see supplemental Figure 1B for corresponding protein levels of IDO and COX2) compared with CD11b+ cells isolated from blood (ascites-isolated, n = 7; control blood-isolated, n = 5). (F) Suppression of CFSE-labeled allogeneic naive CD8+ T-cell proliferation (CD3/CD28 stimulation) in the presence or absence of primary cells or CD11b+ cells isolated from cancer (ie, MDSCs isolated from cancer; n = 7). Percentages indicate the fraction of proliferating granzyme B+CD8+ cells. The gray squares represent the lymphocyte-specific gates used to exclude (CFSE-unlabeled) MDSCs. All data (panels A-F) were confirmed in at least 3 independent experiments. Histograms present data of a single representative experiment with different donors as means ± SD. *P < .05; **P < .01; and ***P < .001.$

PGE₂ redirects DC differentiation and induces CD14⁺CD33⁺CD34⁺ cells with the phenotype and function of monocytic MDSCs isolated from cancer patients. (A) Phenotype of PGE₂-induced CD1a⁻CD14⁺CD80⁻CD83⁻ MDSCs expressing inhibitory molecules ILT2, ILT3, ILT4, PDL-1, but not PDL-2. PGE₂-induced MDSCs express E-prostanoid receptors (labeled with α-EP1–, α-EP3–, sec.Alexa488, α-EP2–, and α-EP4–PE). (B) Expression of immunosuppressive factors IL-10, IDO1, IL-4Rα, and COX2 in PGE₂-induced MDSCs (see supplemental Figure 1A for corresponding protein levels of IDO and IL-10). (C) Immunosuppressive effects of PGE₂-induced MDSCs on allogeneic naive CFSE-labeled CD8⁺ T cells primed by CD3/CD28 and stained for granzyme B. Left panel: Percentages indicate the fraction of proliferating granzyme B⁺ (marker of CTL status) CD8⁺ cells. Right panel: Percentage of proliferating CD8⁺ T cells in the presence of PGE₂-induced MDSCs (PGE₂-d0) and PGE₂-conditioned DCs (PGE₂-d6). (D-F) MDSC phenotype and function of CD11b⁺ cells isolated from cancer ascites. (D) Characterization of cells from cancer ascites either before (left panel) or after (right panel box) isolation of CD11b⁺ cells. Note the high percentage of CD11b⁺ cells (8.9%-50.0%, mean 24.2%, n = 7) within the cancer-infiltrating primary cell population. (E) mRNA levels of IL-10, IDO1, IL-4Rα, and COX2 in CD11b⁺ cells isolated from cancer (see supplemental Figure 1B for corresponding protein levels of IDO and COX2) compared with CD11b⁺ cells isolated from blood (ascites-isolated, n = 7; control blood-isolated, n = 5). (F) Suppression of CFSE-labeled allogeneic naive CD8⁺ T-cell proliferation (CD3/CD28 stimulation) in the presence or absence of primary cells or CD11b⁺ cells isolated from cancer (ie, MDSCs isolated from cancer; n = 7). Percentages indicate the fraction of proliferating granzyme B⁺CD8⁺ cells. The gray squares represent the lymphocyte-specific gates used to exclude (CFSE-unlabeled) MDSCs. All data (panels A-F) were confirmed in at least 3 independent experiments. Histograms present data of a single representative experiment with different donors as means ± SD. *P < .05; **P < .01; and ***P < .001.

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