Figure 3. Extracellular potassium mediated T cell suppression requires intact PP2A activity and is associated with elevations in intracellular potassium.

(a) Selected phosphatase inhibitors from a pharmacologic screen for IFN-γ production of CD8⁺ T cells in the presence of elevated [K⁺]e, depicted as the ratio of IFN-γ expression in Ctrl / elevated [K⁺]e conditions in the presence of indicated phosphatase inhibitors (b) CD8⁺ T cell phosphorylation of pAkt^S473 and pS6^S235/6 10 minutes following TCR cross-linking in the indicated conditions. (c) Compiled analysis of IFN-γ production by retrovirally transduced CD8⁺ Thy1.1⁺ T cells following TCR stimulation in the indicated conditions. (d) Pictorial representation of the resultant intracellular changes in plasma membrane potential (V_m) and intracellular potassium concentration ([K⁺]i) in the presence of elevated extracellular potassium ([K⁺]e). (e) Relative cytoplasmic V_m of CD8⁺ T cells in the indicated conditions represented as relative fluorescence of the voltage-sensitive fluorescent indicator DiSBAC₄(3). (f) Pictorial representation of the resultant intracellular changes in plasma membrane potential (V_m) and intracellular potassium concentration ([K⁺]i) in the presence of the ionophore gramicidin. (g) Relative cytoplasmic V_m of CD8⁺ T cells in the indicated conditions assayed as in (e) with representative flow cytometry in (h). (i) IFN-γ production by CD8⁺ T cells following TCR induced stimulation in the indicated conditions. (j) Representative flow cytometry representing [K⁺]i of CD8⁺ T cells as relative fluorescence of the potassium sensitive dye Asante-Green 4. (k) Relative [K⁺]i of CD8⁺ T cells in the indicated conditions assayed as relative fluorescence of the potassium sensitive dye Asante-Green 4. (l) Representative flow cytometry of cytokine expression by CD8⁺ T cells following TCR stimulation in the indicated conditions with compiled quantification in (m). Error bars represent mean ± SEM. *P < 0.05; **P< 0.01 ***P < 0.001; ****P < 0.0001 between selected relevant comparisons, 2-tailed Student’s t tests (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.