Fig. 3: SIRT2 deficiency augments downstream TCR signaling in T cells.

a, Immunoblots of NFATc2, phospho-S73 and total c-JUN in cytosolic and nuclear fractions of WT and Sirt2^−/− mouse naive CD3⁺ T cells before and after stimulation with 5 µg ml⁻¹ plate-bound CD3 for 30 min. GAPDH and HDAC1 serve as cytosolic and nuclear loading controls, respectively. b, Immunoblot of p-Y202/204 and total ERK1/2 in WT and Sirt2^−/− naive CD3⁺ T cells before and after stimulation with 5 µg ml⁻¹ plate-bound CD3 Abs for 0, 1, 2 and 5 min. GAPDH was used as loading control. c, Representative flow cytometry analysis of p-ERK1/2 (left) and frequencies of p-ERK1/2⁺ CD4 ⁺T cells (left) before and after stimulation with 0.5 µg ml⁻¹ CD3 Ab for 0, 1, 2, 5, 15 and 30 min, in WT or Sirt2^−/− naive CD4⁺ T cells. Each dot represents one mouse (n = 3 mice per group). d–f, Relative Nr4a1 mRNA expression in WT and Sirt2^−/− CD3⁺ T cells freshly isolated (T_N cells) or preactivated for 48 h with 5 µg ml⁻¹ plate-bound CD3 (preactivated T cells) after isolation from the spleen of C57BL/6 mice (d), CD4⁺ T cells freshly isolated (T_N cells), preactivated for 48 h with 10 µg ml⁻¹ OVA peptide (preactivated T cells) or differentiated into T_M-like cells by 5 days of culture with IL-15 (T_M cells) after isolation from the spleen of OT-II mice (e), or CD8⁺ T cells freshly isolated (T_N cells), preactivated for 48 h with 1 µg ml⁻¹ gp100 peptide (preactivated T cells) or differentiated by 5 days of culture with IL-15 (T_M cells) after isolation from the spleen of PMEL mice (f) and analyzed by RT–qPCR before (0 h) and after (2 h) stimulation with 5 µg ml⁻¹ plate-bound CD3 Abs for 2 h. Each dot represents one mouse (n = 3 mice per group). g, Representative flow cytometry analysis of CD62L expression (left) and frequencies of CD62L⁻ T cell subsets (right) in CD4⁺ and CD8⁺ T cells from WT and Sirt2^−/− mice before and after stimulation with 0.25 µg ml⁻¹ CD3 Abs for 90 min. Each dot reprersents one mouse (n = 3 mice per group). h,j, Representative flow cytometry analysis of CD62L expression in CFSE-labeled Sirt2^−/− and CTV-labeled WT T cells preactivated with 0.5 µg ml⁻¹ CD3 Abs for 1 h (h) or left unstimulated (j) before injection into C57BL/6 recipient mice. i,k, Representative flow cytometry analysis (right) and frequencies normalized to WT T cells within each mouse (right) of CFSE⁺ Sirt2^−/− and CTV⁺ WT T cells preactivated as in h (i) or left unstimulated (k) before injection and in the blood, LNs and spleens of recipient mice 1 h after co-transfer transfer (1:1) into C57BL/6 recipient mice. Each dot represents one recipient mouse (n = 5). Data are mean ± s.e.m. P values were determined by two-way analysis of variance (ANOVA) (c–g,i,k). Data are representative of at least two (a,b) and three (c–k) independent experiments.

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