Fig. 3: Aged CD8+ T cells present mitochondrial dysfunction associated with NAD decline.
From: Age-associated nicotinamide adenine dinucleotide decline drives CAR-T cell failure
a, TMRM and MitoTracker green staining in freshly isolated bulk CD8+ T cells from the spleens of young and old mice. TMRM is a cell-permeable dye that accumulates in active mitochondria with intact membrane potential, while MitoTracker green binds to mitochondrial proteins giving a readout of mitochondrial activity and size, respectively (n = 5 biologically independent samples). b, Representative TMRM and MitoTracker green dot plots of data summarized in a. c, EM images of young and old naive CD8+ T cells. Red arrows indicate mitochondrial cristae. Scale bar, 1 μm. d,e, The number of mitochondria per cell (d) and the number of cristae per mitochondria (e) found by EM (n = 3 biologically independent samples). In d, dots represent the number of cells analyzed (n = 20). NS, not significant. In e, dots represent the number of mitochondria analyzed (n = 50). f, Volcano plot representing metabolomic data in young versus old CD8+ T cells (n = 5 biologically independent samples). g, Young (8 weeks old), intermediate (50 weeks old) and old (105 weeks old) CD8+ T cells were activated for 3 days and treated with the NAD precursor NMN (1 mM) for another 2 days, after which mitochondrial activity was assessed by TMRM staining (n = 4 biologically independent samples). h, Young and old CD8+ T cells were activated and expanded until day 7 in the presence of IL-7 and IL-15, after which they further received three rounds of CD3 restimulation every other day to promote an exhausted phenotype. Cells were treated with the NAD-booster NR (1 mM) and levels of the transcription factor TOX were determined on day 12 (n = 3 biologically independent samples). Data are presented as the mean values ± s.e.m. Statistical analysis was performed using an unpaired t-test (a,d,e), two-way ANOVA (g) or paired t-test (h), as appropriate. Statistical analysis of metabolomic data was performed using a two-way ANOVA on log10-transformed data and corrected with the Benjamini–Hochberg method. FC, fold change.
