Figure 2

CCR4 expression in CTLs was induced by antigen-specific TCR stimulation, enhanced by TGF-β1, and suppressed by trametinib. CMV-CTLs were stimulated with HSC-2pp65, HSC-3pp65, HSC-4pp65, and immobilized anti-CD3 mAb. The E:T ratio was adjusted to 1.0 (a,c,d) and 0.5 (e). 10 ng/mL TGF-β1 and 1 μmol/L (a–d) or serial concentration (e) trametinib were added. CCR4 expression in CMV-CTLs was evaluated by flow cytometry on day 2. CCR4 expression in CTLs stimulated by HSC-3pp65 ((a), n = 7, independent donors), by anti-CD3 mAb ((b), n = 7, independent donors), by three different OSCC cell lines ((c), n = 3, independent cell lines) are shown. HLA*A02:01-restricted CMV-CTL derived from HD4 was co-cultured with HLA-mismatched HSC-2pp65 and HLA-matched HSC-3pp65. The respective CCR4 expression is shown in (d). Concentration-dependent effects of trametinib to downregulate CCR4 expression in CTLs co-cultured with HSC-3pp65 without TGF-β1 are shown ((e), n = 7, independent donors). Error bars indicate standard errors. Statistical analysis was performed using Tukey’s multiple comparison test (a–c) or Two-sided Dunnett’s test (e), and p < 0.05 was considered significant. Details of CMV-CTL donors are shown in Supplementary Table S1. Pt. patient, E:T ratio effector to target ratio, Tra trametinib, mAb monoclonal antibody, nM nmol/L, n.s. not significant, HLA human leukocyte antigen.