Figure 4

Tumor bearing conditions down-regulate miR-17-5p expression in T cells. SPCs were harvested from C57BL/6 or STAT6^-/- mice bearing day 15 subcutaneous B16 melanoma (T+) or control non-tumor bearing mice (T-). (A), CD4⁺ and CD8⁺ T cells were isolated by immuno-magnetic bead separation, and evaluated for miR17-5p expression. (B), 1 × 10⁶ CD4⁺ cells from WT mice were briefly stimulated with anti-CD3 mAb for 6 hours. Concentration of IFN-γ secreted in culture media was evaluated by specific ELISA. (C), CD4⁺ T cells were isolated from healthy donor-derived peripheral blood mononuclear cells (PBMC) and stimulated with 5 μg/ml plated anti-CD3, feeder cells (irradiated PBMC) and 100 IU/ml hIL2 in the presence or absence of hIL-4 (10 ng/ml) for 5 days prior to extraction of total RNA. (D), Non-stimulated CD4⁺ and CD8⁺ T cells were isolated by immuno-magnetic beads from PBMC derived from healthy donors (n = 6) or patients with GBM (n = 8) and miR-17-5p expression was analyzed by RT-PCR. Data in (A), (B) and (C), are representative of 2 identical experiments with similar results. Columns represent the mean of triplicates from a single experiment and error bars represent standard deviation. * indicates p < 0.01 and ** indicates p < 0.05 between the two groups using the student t test.