Figure 4. Kcna3 mediated augmented K⁺ efflux lowers intracellular [K⁺], enhances Akt-mTOR signalling, and augments anti-tumour effector function to improve tumour clearance and host survival.

(a) Pictorial representation of the potassium channel K_v1.3 (Kcna3) (b) Representative immunoblot analysis of K_v1.3 protein abundance in CD8⁺ Pmel-1 cells following retroviral transduction with Ctrl (Empty-Thy1.1) or Kcna3-Thy1.1 constructs. (c,d) Thy1.1⁺ Pmel-1 CD45.1⁺CD8⁺ TIL 6-8 days following transfer into B16 melanoma-bearing mice were analysed for indicated phospho-residues (c) or in vivo IFN-γ production 6 hours after injection with Brefeldin A (d). (e) Relative cytokine expression of human TIL originating from the indicated histology following TCR stimulation in the indicated conditions. (f,g) Analysis of intracellular [K⁺] (f) and representative flow cytometry for the expression of the indicated cytokines (g) of CD8⁺ Thy1.1⁺ T cells following transduction with retrovirus expressing Ctrl, Kcna3, or Kcna3_PD Thy1.1⁺ constructs. (h,i) Rates of tumour growth (h) and host survival (i) represented over time following receipt of Pmel-1 CD8⁺ T cells transduced as in (f,g). 2-tailed Student’s t tests (c-e), 2-way ANOVA (f), Wilcox rank-sum analysis (h), and Log-rank of Kaplan-meier survival estimates (i). Error bars represent mean ± SEM. *P < 0.05; **P< 0.01 ***P < 0.001; ****P < 0.0001 between selected relevant comparisons, additional [K⁺]e equal to 50mM in (e). (c) five mice per group (d) seven mice per group (e) each symbol represents the mean of three culture replicates per patient per data point (f) three technical replicates per data point (h,i) at least ten mice per group, representative of (b) three, (c-i) two independent experiments.