Figure 5.

Trametinib + anti–PD-1 therapy unleashes the immune system against iCCA tumors. (A) Representative contour plot of CD4⁺ and CD8⁺ gating strategy of hepatic lymphocytes of orthotopic SB1 tumor-bearing mice after treatment. Experimental set-up is shown in Figure 4A. Percentage of hepatic (B) CD44^highCD62L^low CD8⁺ and (C) CD4⁺ effector memory T cells after intrahepatic SB1 injection (n = 5 per group). Experimental set-up is shown in Figure 4A. (D) Representative contour plot and frequencies of CD44^highCD62L^low CD8⁺ effector memory T cells of hepatic lymphocytes of orthotopic SB1 tumor-bearing mice after treatment (gating strategy CD3⁺CD8⁺). Experimental set-up is shown in Figure 4A. (E) Percentage and (F) representative contour plot and frequencies of CD107⁺CD8⁺ T cells of hepatic lymphocytes of orthotopic SB1 tumor-bearing mice after treatment (gating strategy CD3⁺CD8⁺) after ex vivo stimulation for 4 hours (n = 5 per group). Experimental set-up is shown in Figure 4A. (G) Percentage and (H) representative contour plot and frequencies of IFNγ⁺CD8⁺ T cells of hepatic lymphocytes of orthotopic SB1 tumor-bearing mice after treatment (gating strategy CD3⁺CD8⁺) after ex vivo stimulation for 4 hours (n = 5 per group). Experimental set-up is shown in Figure 4A. (I) Percentage and (J) representative contour plot and frequencies of CD107⁺CD4⁺ T splenocytes from SB1 flank-tumor-bearing mice after ex vivo stimulation with SB1 tumor cells for 5 hours (n = 5 per group). Group set-up is shown as in Figure 3A. Data represent means ± SD. ∗P < .05, ∗∗P < .01; Student t test. aPD-1, anti-Programmed cell death protein-1; FSC-A, forward scatter area; FSC-H, forward scatter high; L/D, live/dead; SSC,side scatter; T, trametinib.