Figure 2.

(A) Peritoneal macrophages from healthy mice and MDSC from tumor-bearing mice were stained with mAb to CD11b, F4/80, Gr1, Ly6C, and/or Ly6G and analyzed by flow cytometry. MDSC from WT, IL-10^−/−, and IL-6^−/− BALB/c mice with 4T1 tumors were assayed for their ability to suppress the antigen-driven activation of peptide-specific, MHC-restricted, transgenic CD4⁺ (DO11.10) and CD8⁺ (Clone 4) T cells. (B–D) 4T1-induced MDSC and peritoneal macrophages (Mac) from WT, IL-10^−/− (10^−/−), or IL-6^−/− (6^−/−) BALB/c mice were cocultured, and supernatants were assayed for IL-10, IL-6, and NO. (B) Macrophages enhance MDSC IL-10, and MDSC decrease macrophage IL-6. (C) IL-6 decreases MDSC IL-10. (D) IL-10 production by MDSC decreases macrophage IL-6 and TNF-α and increases macrophage NO. (E) Neutralizing antibodies to IL-10 prevent the down-regulation of macrophage IL-6 and IL-12. (F) Exogenous IL-10 decreases MDSC and macrophage TNF-α, decreases macrophage IL-6, and enhances macrophage NO. Macrophages or MDSC were cultured with IL-10 or denatured IL-10. (G) Macrophages activate STAT3 in response to IL-10. Macrophages (left) were cultured for 5 min in the presence or absence of exogenous IL-10 (middle) or with supernatants (media) from MDSC-macrophage cocultures (right), subsequently fixed and permeabilized, and then stained for F4/80, CD11b, and pSTAT3. (H) Macrophages and MDSC express the receptors for IL-10 and IL-6, respectively. (A–H) Data are from one of two, 30, three, five, four, three, two, and two experiments, respectively. Statistical significance was determined by (A–E) Tukey's HSD test and (F) the Mann-Whitney test. Different lower case letters above each value indicate that those values are statistically, significantly different; values that share the same lowercase letter are not statistically, significantly different.