Epithelial loss of TβRII results in increased Th17 cell development.

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Epithelial loss of TβRII results in increased Th17 cell development. Epithelial loss of TβRII results in increased Th17 cell development. A, data from ELISA analysis of IL-6, IL-23, and TGF-β1. Cultured carcinoma cells (1 × 105/mL) were incubated for 24 hours in DMEM + 5% FBS. Tumor explants were prepared as described in the Methods. CD45+ and CD45− cells were isolated by CD45 magnetic microbeads and incubated for 24 hours in 1 × 106/mL in RPMI + 10% FBS. CM, conditional medium of carcinoma cells; TE, tumor explant. B, quantitative data of CD4+ T cells isolated from tumor tissue of PyMT/Tgfbr2fl and PyMT/Tgfbr2KO mice on day 9 of tumor progression. Before flow cytometry analysis, cells were incubated for 6 hours with CD3/CD28 beads (left). Isolated CD4+ cells from tumor tissue of PyMT/Tgfbr2fl and PyMT/Tgbfr2KO mice were incubated for 24 hours with CD3/CD28 microbeads and then the levels of IL-17A/F in conditional medium were measured by ELISA following the manufacturer's protocol (right). Data correspond to the mean ± SEM of 3 individual mice from 3 experiments. C, qPCR of TGF-β1, IL-23 (p19), and IL-6 from sorted TAMs (left) and MDSCs (right) from tumor tissue of PyMT/Tgfbr2fl and PyMT/Tgfbr2KO mice on day 9 of tumor progression. Cells were sorted by FACSAria. D, MDSCs (CD11b+Gr1+) were isolated from spleen of MMTV-PyMT FVB tumor-bearing mice by magnetic microbeads (Gr1) and then incubated for 24 hours with IL-17 (10 ng/mL), then total RNA was isolated and expression of genes was analyzed by qRT-PCR. The fold change in expression by treated vs. untreated cells is shown (left). Monocytes were differentiated from bone marrow of naïve FVB mice in presence of macrophage colony-stimulating factor (10 ng/mL). Suspension cells were removed and attached cells were harvested by trypsin and analyzed. One portion of cells was analyzed by flow cytometry with the CD11b marker. The remaining cells were incubated for 24 hours with IL-17 (10 ng/mL), and then total RNA was isolated and expression of genes was analyzed by qRT-PCR. The -fold change in expression by treatment over untreated cells is shown (right). Data correspond to the mean ± SEM of 3 individual mice from 2 experiments. E, CD11b+F4/80+Gr1− cells (TAMs) and CD11b+Gr1+ cells (MDSCs) were sorted by FACSAria from tumor tissue of PyMT/Tgfbr2fl and PyMT/Tgfbr2KO mice on day 9 of tumor progression. Total RNA was isolated immediately after sorting and expression of genes was analyzed by qRT-PCR. Data correspond to the mean ± SEM of 2–3 individual mice from 3 experiments. PyMT/Tgfbr2fl is Tgfbr2FL, and PyMT/Tgfbr2KO is Tgfbr2KO. ND, not detected. *P < 0.05; **P < 0.01. Sergey V. Novitskiy et al. Cancer Discovery 2011;1:430-441 ©2011 by American Association for Cancer Research