Fig. 4: TCR activation-mediated KCa3.1 methylation regulates T cell effector function.

a,b, Representative images (a) and quantification (b) of methylarginine (meArg) in CD8⁺ T cells activated with anti-CD3/28 Dynabeads (dark gray masked) in 0.1, 0 and 0.03 mM Met for 30 min, stained with pan-meArg (red, with arrows), Hoechst (blue) and Phalloidin (green) (each circle represents one cell; n = 26 cells per group. Scale bar, 10 μm. c, Venn diagram of enriched meArg proteins at 30 min postactivation of T cells in 0.1 mM versus 0 mM or 0.1 mM versus 0.03 mM Met, by anti-CD3:IgG crosslinking for 30 min, as identified by TMT-MS. d, Heatmap of TMT-MS-identified proteins (P < 0.05) with enriched meArg in T cells activated in 0.1 mM versus 0 mM or 0.03 mM Met as in c (n = 2 biological replicates per group). e, Predicted interaction of KCa3.1 monomer with CaM as unmethylated (left), SDMA (center) and ADMA (right). The CaM-binding pocket is represented as surface rendering with interacting amino acids as sticks and R352 as ball and sticks. f, MD simulation analysis. Analysis of variations of distance of salt bridge between CaM E84 and demethylated R352 WT (purple), SDMA R352 (green) and ADMA R352 (blue) over the course of simulation time (averaged across all four monomers, three MD trials). g, Representative plot of Indo-1 analysis of Ca²⁺ flux in CD8⁺ T cells activated with anti-CD3 and anti-CD28 by anti-hamster IgG crosslinking in Ca²⁺-free Ringer solution supplemented with either 0.1 mM or 0.03 mM Met and treated with either with DMSO or 1 μM TRAM-34 (n = 5 biological replicates per group). h,i, AUC (h) and maximum peak signal (i) from g normalized to 0.1 mM Met (n = 5 biological replicates per group). j, Tumor growth of B16-OVA tumors in Rag1^−/− mice after transfer of OT-I CD8⁺ T cells activated in 0.1 mM or 0.03 mM Met with either DMSO or 1 μM TRAM-34 for 30 min and cultured for 24 h (n = 5 mice per group). Data are mean ± s.d. Boxplots shows minimum and maximum value with median as center. Unpaired two-tailed Student’s t-test (b), paired two-tailed Student’s t-test (h,i), two-way ANOVA (j).

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