Fig 2: SIRT2 inhibition ameliorates T cell activation in the lungs and spleen of infected mice.Cells isolated from the lungs of control and AGK2-treated Mtb-infected C57BL/6 animals were subjected to surface staining (CD3-Pacific Blue, CD4-PE, CD8-APC/Cy7, CD69-FITC and CD25-APC) followed by FACS analysis. (A) Gating strategy and representative dot plots. Scatter plots depicting the percentage of CD4+, CD4+CD69+, CD4+CD25+, CD8+, CD8+CD69+ and CD8+CD25+ T cells in the (B–G) lungs and the (H–M) spleen of infected mice. The experiment was performed twice with five mice in each group. Data is represented as mean ± SD (n = 5). **p<0.005, ***p<0.0005.Figure 8—source data 1.SIRT2 inhibition increases T cell activation in vivo.Source data for Figure 8B–M.

Fig 3: SIRT2 inhibition activates macrophages and T cells to induce pro-inflammatory response.(A) Schematic representation of the experimental plan (for details see Materials and methods). 48 hr post co-culture, macrophages were stained with CD11b (APC/Cy7), CD11c (APC), CD80 (FITC) and MHC-II (PE) followed by flow cytometry analysis. (B) Gating strategy employed and representative overlay plots to depict macrophage activation. Expression of (C) CD11b, (D) CD11c, (E) CD80 and (F) MHCII on the surface of peritoneal macrophages. (G) Gating strategy for T cell activation. Splenocytes were stained for surface markers CD3 (Pacific Blue), CD4 (PE), CD8 (APC/Cy7), CD69 (FITC) and CD25 (APC) followed by flow cytometry. Percentage of (H) CD4+, (I) CD4+CD69+, (J) CD4+CD25+, (K) CD8+, (L) CD8+CD69+ and (M) CD8+CD25+ T cells. For cytokine analysis, splenocytes were stained with CD3 (Pacific Blue), CD4 (PE), CD8 (APC/Cy7), IFNγ (APC) and Il17 (PE/cy7). (N and O) Intracellular cytokines (IFNγ and IL17) expressed by CD4+ T cells. (P and Q) CD8+ T cells expressing IFNγ and IL17. (R) CFU at 48 hr post co-culture. Combined data from two independent experiments performed in triplicates is shown. Each bar represents mean ± SD (n = 6). Statistical analysis performed using one-way ANOVA followed by Tukey post hoc test. *p<0.05, **p<0.005, ***p<0.0005.Figure 4—source data 1.SIRT2 inhibition leads to the activation of macrophages and T cells upon Mtb infection.Source data for Figure 4C–F and H–R.

Fig 4: T cell subsets in aged Optn470T were comparable to WT male mice. The gating strategy in mouse spleens is shown for a representative two-year-old mouse (A). Population frequency (%) and absolute cell numbers (No) are shown for one- and two-year-old WT and Optn470T as follows: frequency of T cells (B), CD8+ and CD4+ T cells (C), CD8+ naïve (TN), central memory (TCM) and effector memory (TEM) (D), and CD4+ TN, TCM and TEM (E); numbers of T cells (F), CD8+ and CD4+ T cells (G), CD8+ TN, TCM and TEM (H), and CD4+ TN, TCM and TEM (I). Staining for the indicated cytokines upon PMA/ionomycin stimulation is shown as frequencies and MFI in CD8+ (J,K) and CD4+ (L,M) T cells. The gating strategy for Treg cells in mouse spleens for representative two-year-old mice is shown. Functional Tregs were gated as CD4+FOXP3+CD25+, and non-functional as CD4+FOXP3+ CD25- cells (N). The graph shows the MFI of FOXP3 in Optn470T normalized to one- and two-year-old WT mice in functional Tregs (O). Treg subsets are shown as frequency (P) and numbers (Q). The data from 6–7 one-year- and two-year-old WT and Optn470T mice are shown as means ± SEM and analysed by two-way ANOVA ((B,C,D): TN; (E,F,G,H): TN and TEM; (I,J,L,P) and (Q), Kruskal–Wallis test ((D): TCM, TEM; (H): TCM) and Student’s t-test (K,M,O): *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 5: Protein quality control in ß-selected thymocytes.(A) Schematic of labeling and detection of nascent protein with OP-Puro. OP-Puro (O-propargyl puromycin) is a cell-permeable puromycin analog that is incorporated into the C-terminus of newly synthesized peptide chain. Fluorophore conjugated with Alexa Fluor 647 was then attached to OP-Puro through a copper-catalyzed click chemistry reaction between alkyne and azide group, which quantifies protein synthesis by fluorescence intensity. (B) Representative histogram (left) and quantification (right) of OP-Puro incorporation in different thymocyte subsets from 8-week-old wild-type mice. FMO represents AF647 control which is the background from the click chemistry in the absence of OP-Puro. MFI, mean fluorescence intensity. n = four mice. (C) Quantification of tetraphenylethene maleimide (TMI) fluorescence in different thymocyte subsets from 8-week-old wild-type mice. n = seven mice. (D) Quantitative RT-PCR analysis of ERAD (Sel1l) and UPR-related (Xbp1, Hspa5(Bip), Dnajb9, Ddit3 (Chop), Atf4) genes expression in different thymocyte subsets from 6-week-old wild-type mice. Data are presented relative to Actb; n = three mice. (B–D), ETP: early T lineage precursor (Lin- CD4- CD8- CD44+ CD25- CD117+); DN2: double negative two thymocytes (Lin- CD4- CD8- CD44+ CD25+); DN3: double negative three thymocytes (Lin- CD4- CD8- CD44- CD25+); DN4: double negative four thymocytes (Lin- CD4- CD8- CD44- CD25-); DP: double positive thymocytes (Lin- CD4+ CD8+); SP4: CD4 single positive thymocytes (Lin- CD4+ CD8-); SP8: CD8 single positive thymocytes (Lin- CD4- CD8+). Results are shown as mean ± s.d. The statistical signi?cance was calculated by one-way ANOVA with Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not signi?cant.Figure 1—source data 1.Excel file containing numerical values shown in Figure 1.