Fig. 1: SIRT2 deficiency enhances Ca²⁺ flux and proximal TCR signaling in T cells.

a, Ca²⁺ flux in naive CD4⁺ (left) and CD8⁺ (right) T cells isolated from the spleen of WT and Sirt2^−/− mice, loaded with Indo-1 AM and 0.5 µg ml⁻¹ biotinylated CD3 Abs. Baseline fluorescence ratio was recorded for 60 s before simulation with 2.5 µg ml⁻¹ streptavidin (black arrow). Ionomycin was added later (gray arrow) as a control of maximal Ca²⁺ release. n = 3 mice per group. b, Ca²⁺ flux in WT or Sirt2^−/− CD8⁺ PMEL T cells treated as in a and stimulated with gp100-loaded or empty EL4 cells. n = 3 mice per group. c, Ca²⁺ flux in WT or Sirt2^−/− CD4⁺ T cells treated as in a and stimulated with a concentration range (0.015, 0.03, 0.06, 0.125 and 0.25 µg ml⁻¹) of biotinylated CD3 Abs. n = 3 mice per group. d, Ca²⁺ flux in WT or Sirt2^−/− CD4⁺ T cells treated as in a and stimulated with a concentration range (1, 5 and 10 µg ml⁻¹) of biotinylated CD3 Abs. n = 3 mice per group. e, Ca²⁺ flux in preactivated WT or Sirt2^−/− CD4⁺ and CD8⁺ T cells prestimulated with 5 µg ml⁻¹ plate-bound CD3 for 48 h, and then re-stimulated with 0.5 µg ml⁻¹ biotinylated CD3 as in a. n = 3 mice per group. f, Ca²⁺ flux in WT or Sirt2^−/− CD4⁺ OT-II (left) and CD8⁺ PMEL (right) T cells cultured with IL-15 for 5 days following 72 h OVA or gp100 stimulation to generate T_M-like cells, and then treated as in a with 2.5 µg ml⁻¹ biotinylated CD3 Abs. n = 3 mice per group. g, Flow cytometry analysis of p-Y394 LCK (left) and frequency of p-Y394 LCK⁺ CD4⁺ T cells (right) before and after stimulation of WT or Sirt2^−/− naive CD4⁺ T cells with 0.5 µg ml⁻¹ CD3 Ab for 0–30 min. Each dot represents one mouse (n = 3 mice per group). Data are mean ± s.e.m. h, Immunoblot analysis of p-Y394 and total LCK in WT and Sirt2^−/− naive CD3⁺ T cells before and after stimulation with plate-bound CD3 Abs for 0 min and 5 min. i, Immunoblot analysis of phosphorylated and total forms of ZAP70 and PLCγ1 in WT and Sirt2^−/− CD4⁺OT-II T cells preactivated with OVA peptide for 48 h, before and after stimulation with plate-bound CD3 Abs for 0, 1, 2 and 5 min. j, Immunoblot analysis of phosphorylated and total forms of LCK, ZAP70 and LAT in Sirt2^−/− T cells transduced with empty or SIRT2-expressing lentivector, before and after stimulation with plate-bound CD3 Abs for 0, 1, 2 and 5 min (top) and immunoblot of SIRT2 expression in Sirt2^−/− T cells transduced with vehicle, empty lentivector or SIRT2-expressing lentivector. In h–j, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as loading control. Data are representative of two (b–e,j) and three (a,f–i) independent experiments.

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