Fig 2: The effect of SIRT5 on the effector function, mitochondrial function and transcription factor expression of CD8+ T cells from the spleens of co-transferred mice. (A) Representative dot plots of IL-2, TNFα, and IFNγ expression levels of OT-1 cells from the spleen tissues by flow cytometry in vitro restimulation. The transferred mice are sacrificed on day 25 after secondary LM-OVA infection. (B) Statistical analysis of OT-1 cell ratio of cytokine experiment. Mean ± SD (n = 6), Student’s t-test. (C) Statistical analysis of relative MTG and TMRE MFI. Mean ± SD (n = 6), Student’s t-test, **: p < 0.01. (D) Representative histograms of Tcf1 and T-bet protein expression level of Sirt5+/+ and Sirt5−/− OT-1 cells from the spleens. (E) Statistical analysis of Tcf1 and T-bet MFI. Mean ± SD (n = 6), Student’s t-test.

Fig 3: Restoration of miR-181a expression in old naive CD4 T cells by inducing TCF1 activity.a TCF7 transcripts in naive CD3 + CD4 + CD45RO- T cells were quantified by qPCR. Results for 20–35 (n = 19) and 65–85 (n = 18) year-old individuals are shown as box plots of TCF7 transcripts relative to ACTB. b TCF1 protein levels in CD3 + CD4 + CD45RO- naive T cells from seven young and seven old adults are shown as Western blots (left) and box plots of relative densities (right). Uncropped Western blots in Supplementary Figure 4. All samples in the blot derive from the same experiment and were processed in parallel. c TCF1 expression levels in naive T cells from eight young and eight old adults are shown as representative histograms (left) and summary box plots (right). d Naive CD4 T cells from old individuals were treated with GSK3β inhibitors 6-bromoindirubin-3-oxime (BIO) or SB216763 for 48 h. TCF7 transcripts quantified by qPCR are shown as mean±SEM (n = 3). e BIO (1 µM) or SB216763 (5 µM) were added to HEK293T cells transfected with pri-miR-181a enhancer reporter constructs 12 h after transfection. Reporter activities after 48 h are shown as mean±SEM (n = 3). f, g Naive CD4 T cells from seven young and seven old individuals were cultured with DMSO solvent control or BIO (f, 1 µM) or SB216763 (g, 5 µM) for 48 h. Pri-miR-181a/b1 transcripts were quantified by qPCR. Comparisons were done by two-tailed unpaired t test in a, b, c, f, and g; or by one-way ANOVA with post-hoc Tukey test in d, e. Significance levels are indicated as *P < 0.05, **P < 0.005, ***P < 0.0001.

Fig 4: Model of TCF1-mediated regulation of miR-181a dependent on GSK3ß.T cell aging is associated with a loss of miR-181a expression due to reduced TCF1 expression (left). Reduced miR-181a expression contributes to impaired T cell function by activating negative regulatory signaling pathways by miR-181a targets, e.g., ERK dephosphorylation by DUSP6. Inhibition of GSK3ß increases the expression of TCF1 and its target miR-181a in T cells from old adults, thus enhancing downstream T cell activation and proliferation (right).

Fig 5: The effect of SIRT5 on the effector function, mitochondrial function, and transcription factor expression of CD8+ T cells from the spleens of transferred mice. (A) Representative dot plots of IL-2, TNFα, and IFNγ expression levels of OT-1 cells from the spleen tissues by flow cytometry in vitro restimulation. The transferred mice are sacrificed on day 34 after primary LM-OVA infection. (B) Statistical analysis of OT-1 cell ratio of cytokine experiment. Mean ± SD (n = 9), Student’s t-test. (C) Statistical analysis of MTG and TMRE MFI. Mean ± SD (n = 9), Student’s t-test, *: p < 0.05. (D) Representative histograms of Tcf1 and T-bet protein expression level of Sirt5+/+ and Sirt5−/− OT-1 cells from the spleens. (E) Statistical analysis of Tcf1 and T-bet MFI. Mean ± SD (n = 9), Student’s t-test.