Fig. 4: Restoration of NAD levels rescues functionality of aged CAR-T cells in vivo.
From: Age-associated nicotinamide adenine dinucleotide decline drives CAR-T cell failure
a, Levels of CD38 in bulk CD8+ T cells upon isolation or 3 days after activation (n = 5 biologically independent samples). b, Representative TMRM histograms of CD38hi and CD38low population in young and old CD8+ T cells 3 days after activation. c, Proportion of TMRMhi cells in CD38hi and CD38low populations (n = 3 biologically independent samples). d, NAD/NADH ratio on day 7 in old CAR-T cells expanded with IL-7 and IL-15 and treated with NMN and/or 78c, a specific inhibitor of the NADase enzymatic activity of CD38 (n = 3 biologically independent samples). e, OCR on day 7 in old CAR-T cells expanded with IL-7 and IL-15 and treated with NMN and/or 78c were measured using a Seahorse XFe96 Analyzer. During this assay, mitochondrial fitness was assessed upon the sequential addition of oligomycin (oligo, adenosine triphosphate synthetase inhibitor), FCCP (mitochondrial membrane uncoupler) and rotenone + antimycin A (Rot/AA, complex I and III inhibitors, respectively). f,g, Further analysis of the recovery of SRC (f) and mitochondrial size (g) of old CAR-T cells treated with NMN and 78c. Seahorse data displayed in f are representative of three independent experiments (n = 6 technical replicates). For g, n = 3 biologically independent samples. h, CD62L levels within TCM populations (n = 5 biologically independent samples). In i–k, young and old CAR-T cells treated with or without NMN and 78c were adoptively transferred into mice bearing B16-HER2 tumors. i,j, Tumor growth (i) and tumor size at last time point (day 30) (j) (n = 9 young, n = 8 old and n = 8 old 78c + NMN). k, Number of transferred T cells (CD45.1+) found in spleen 32 days after tumor engraftment (n = 7 mice). In l–n, young and old CAR-T cells treated with or without NMN and 78c were adoptively transferred into mice bearing EL4-mCD19 tumors (n = 8 mice per group). l,m, Tumor growth (l) and tumor size at last time point (day 17) (m). n, Number of transferred T cells (CD45.1+) found in spleen 17 days after tumor engraftment. Data are presented as the mean values ± s.e.m. Statistical analysis was performed using an unpaired t-test (a,k,n), two-way ANOVA (c) or one-way ANOVA with multiple comparisons (d–m), as appropriate.
