Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-β Production and Th17 Differentiation
Fig 5
IL-17 production via direct interaction of CD40 and CD40L increases STAT3 activation and the proliferation of MDA-MD231 cells.
(A) MDA-MB231 cells and activated T cells were directly co-cultured at the ratio of 1:5 for 24 hrs in the presence of anti-IL-17 neutralizing antibody or anti-CD40 neutralizing antibody (2 μg/ml/each) on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [3H]-thymidine, cells were culture for another 18 hrs. And the proliferation of cells was measured as described in Materials and Methods. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments. (B) MDA-MB231 cells were cultured in the presence of recombinant IL-17 (rIL-17, 50 ng/ml) for 15, 30 and 60 min. In addition, cells were cultured with direct co-culture supernatant of MDA-MB231 cells and activated T cells in the presence or absence of anti-IL-17 neutralizing antibody (nAb). And then, the activation of STAT3 was examined by western blot as described in Materials and Methods. Lane 1: Control, Lane 2: rIL-17 (50 ng/ml), Lane 3: Direct co-culture supernatant of MDA-MB231 cells and activated T cells, Lane 4: Direct co-culture supernatant of MDA-MB231 cells and activated T cells with IL-17 nAb, Lane 5: Indirect co-culture supernatant of MDA-MB231 cells and activated T cells. β-actin was used as a loading control. Result is the representative of three independent experiments. (C) MDA-MB231 cells and activated T cells were directly co-cultured at the ratio of 1:5 for 24 hrs in the presence of 20 μM of AG490 (STAT3 inhibitor) or anti-IL-17 nAb (2 μg/ml) on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [3H]-thymidine, cells were culture for another 18 hrs. Direct co-culture supernatant of CD40-non expressing breast cancer cell line, Hs578T and activated T cells was used as a negative control. The proliferation of cells was measured as described in Materials and Methods. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments.