Supplementary Materials3. culture replicates. Representative of (e-g) three (h,i,j) two impartial experiments. We next asked whether elevated [K+]e affects T cell function. We found a striking dose-dependent suppression of TCR-induced cytokine production by isotonic elevations in [K+]e (Fig. 1e,f). Elevated [K+]e acted impartial of tonicity, with other monovalent and divalent ions or inert osmolytes failing to induce comparable suppression (Fig 1f,g and Extended Data 1e-h). Elevated [K+]e functioned to acutely suppress T cell activation across a range of signal strengths (Extended Data 1i), in the presence or absence of co-stimulation (Extended Data 1j), in a nonredundant fashion to tumour-associated co-inhibitory signals (Fig. 1h,i and Extended Data 2a-b), in CD4+ TH1 and TH17 effector subtypes (Extended Data 2c,d), and had no effect on cellular viability (Extended Data 2e). We next isolated endogenous human neoantigen-specific TIL, identified as likely mediators of immunotherapy-induced tumour clearance9,16, and found IFN- production by these cells in response to their cognate neoepitope to be significantly attenuated by elevated [K+]e (Fig. 1j and Extended Data 2f,g). Elevated [K+]e also led to suppression of target-specific IFN- production by T cells genetically engineered with a cancer-germline antigen specific TCR17(Extended Data 2h). Thus, our data suggests that elevated [K+]e acutely limits the function of mouse and human T cells. To understand the basis for this suppression of effector function, we explored the effect of elevated [K+]e around the molecular events Talsaclidine driven by TCR engagement. To this end, we briefly activated FACS-purified murine CD8+ T cells in the presence or absence of elevated potassium and found that elevated [K+]e significantly restrained the expression of transcripts induced by TCR stimulation (Fig. 2a,b). Furthermore, gene-set enrichment analysis indicated that elevated [K+]e suppressed Talsaclidine genes induced by TCR signalling, NF-B activation, escape from anergy, adaptive immune response, and cytokine pathways (Supplementary Information 1). Collectively, these data suggest that intratumoural cell death produces elevated [K+]e concentrations which act to suppress TCR-driven effector programmes. Open in a separate window Physique 2 Extracellular potassium inhibits TCR induced transcripts and function by suppressing Akt-mTOR phosphorylation(a) Pie chart representing proportional subpopulations of all transcripts following 2h re-stimulation of purified CD8+ T cells with anti-CD3/28 (b) Volcano plot of TCR induced genes briefly re-stimulated with anti CD3/28 in the indicated conditions. (c) TCR cross-linking induced calcium flux of CD8+ cells as measured by Fluo3 / FuraRed fluorescence in the indicated conditions. (d) Representative phosflow cytometry plots following TCR cross-linking in the indicated conditions. (e) Immunoblot analysis of the indicated phospho-residues in CD8+ T cells following TCR cross-linking (f) Quantitative phosflow analysis of cells activated as in (c) and (d) with representative flow cytometry in (g). (h) Quantification of the indicated phosphotidylinositol species in CD8+ T cells activated via TCR cross-linking in the indicated conditions. Error bars represent mean SEM. 0.05; ** 0.001; **** 0.0001 between selected relevant comparisons, 2-way ANOVA, Rabbit Polyclonal to Fyn (phospho-Tyr530) (c-h) where noted additional Talsaclidine [K+]e equal to 40mM Talsaclidine (a,b,c) three biological replicates (d,f) three technical replicates per data point (h) three experimental replicates with pooled analysis displayed, (d-g) representative of at least three independent experiments. The observation that elevated [K+]e acutely suppressed TCR-driven transcriptional events led us to inquire whether [K+]e could affect TCR-induced signal transduction pathways. Given the role of [K+]e in regulating plasma membrane potential18,19, we initially hypothesized that K+ acted to suppress TCR activation via induction of cellular membrane depolarization (increased 0.05; ** 0.001; **** 0.0001 between selected relevant comparisons, 2-tailed Students assessments (a-m), where noted additional [K+]e equal to 40mM, (a,c,i,l,m) at least three culture replicates per data point (e,g,h,j,k) three technical replicates per data point, representative of at least (a,b,c,m) two or (e,g,i,h,l,k) three or greater independent experiments. Moreover, OA reversed the hypophosphorylation of Akt and S6 caused by elevated [K+]e (Fig. 3b and Extended Data 5a) in addition to restoring effector function (Fig. 3c and Extended Data 5b)..