Figure 3.

IL-31 promotes a cytotoxic T cell memory phenotype and inhibits MDSC motility. (A) CD8⁺ T cells isolated from naïve mice were co-cultured with Gr1⁺ MDSCs isolated from PyMT tumors. T cells were activated with soluble anti-CD3 and anti-CD28. Cultures were then supplemented with 100 ng/mL IL-31 or left untreated. After 4 days, the activation and memory phenotype of CD8⁺ T cells were evaluated by flow cytometry. (B) The expression of CD44 and CD62L was evaluated in PMN-MDSCs isolated from peripheral blood of mice implanted with PyMT-ev or PyMT-IL-31 tumors. The evaluation was performed by flow cytometry using MFI. (C, D) The motility of MDSCs was measured by seeding one million splenocytes from naïve C57BL/6 mice (n=4 biological repeats) on a 3 µm insert containing SF media in both upper and lower compartments. The cells were treated with 100 ng/mL IL-31 or left untreated (control). After 18 hours, the cells in the lower compartment were collected, stained for Ly6G and Ly6C, and counted by flow cytometry. A summary graph is shown (C) followed by representative flow cytometry dot-plots (D). Statistical significance was assessed by unpaired two-tailed t-test. Statistical significance of MDSC motility was assessed by paired t-test. Significant p values are shown. CTL, cytotoxic T lymphocytes; IL-31, interleukin-31; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; PMN, polymorphonuclear; SF, serum free media.