Figure 4. miR-200c promotes CTL survival and effector function in vivo.

A 1:1 mixture of miR-200c and miR-Scr OT1 CTLs was transferred into B16OVA tumor bearing mice. At various time points, CTLs were extracted and analyzed by flow cytometry. (A) Top left, representative flow cytometry plot showing TNF and IFN-γ expression in the indicated tumor-infiltrating CTLs, extracted 1 week after infusion and restimulated with PMA and ionomycin. Right and below, quantification of TNF⁺ (top right), IFN-γ⁺ (bottom left), and TNF⁺IFN-γ⁺ (bottom right) tumor infiltrating CTLs from unstimulated (un) and PMA and ionomycin stimulated (stim) samples is shown. n = 3 mice per group. (B) Above, representative flow cytometry plot showing TCF1 and T-bet expression in tumor infiltrating CTLs, extracted 1 week after infusion into tumor bearing mice. Below, quantification of TCF1 (left) and T-bet (right) expression in tumor infiltrating CTLs is shown. n = 3 mice per group. (C) Flow cytometric analysis of exhaustion markers in miR-Scr or miR-200c OT1 CTLs, extracted 2 weeks after infusion into B16OVA tumor bearing mice. Quantification of PD-1 (left), LAG3 (middle), and TIM3 (right) expression in CTLs extracted from the tumor and spleen (sp) is shown. n ≥ 6 per group for PD-1 and LAG3, n = 4 per group for TIM3. (D) β-catenin (βcat) abundance in CTLs expressing the indicated miRs was assessed by immunoblot using actin as a loading control. All error bars denote SEM. ns, not significant, *P ≤ 0.05, and **P ≤ 0.01, calculated by Student’s t test. Data in (C) were pooled from two independent experiments. All other data are representative of at least 2 independent experiments.