Metabolic reprogramming of human CD8+ memory T cells through loss of SIRT1

Abstract

The expansion of CD8⁺CD28^- T cells, a population of terminally differentiated memory T cells, is one of the most consistent immunological changes in humans during aging. CD8⁺CD28^- T cells are highly cytotoxic, and their frequency is linked to many age-related diseases. As they do not accumulate in mice, many of the molecular mechanisms regulating their fate and function remain unclear. In this paper, we find that human CD8⁺CD28^- T cells, under resting conditions, have an enhanced capacity to use glycolysis, a function linked to decreased expression of the NAD⁺-dependent protein deacetylase SIRT1. Global gene expression profiling identified the transcription factor FoxO1 as a SIRT1 target involved in transcriptional reprogramming of CD8⁺CD28^- T cells. FoxO1 is proteasomally degraded in SIRT1-deficient CD8⁺CD28^- T cells, and inhibiting its activity in resting CD8⁺CD28⁺ T cells enhanced glycolytic capacity and granzyme B production as in CD8⁺CD28^- T cells. These data identify the evolutionarily conserved SIRT1-FoxO1 axis as a regulator of resting CD8⁺ memory T cell metabolism and activity in humans.

Figure 1.

SIRT1 levels are down-regulated in human CD8⁺CD28^– T cells. (A) Sorting strategy for CD8⁺ naive, a CD28⁺-expressing T_CM/T_TM/T_EMRA pool, and CD28⁻ T_EM/T_EMRA cells from a healthy donor based on surface markers CD3, CD8, CD28, CCR7, and CD45RA. (B–D) SIRT1 expression was assessed by Western blot (B and C, n = 9), and qRT-PCR was normalized to RPL13A (n = 7; paired one-way ANOVA; D). (E) SIRT6 and SIRT7 expression was measured by Western blot (n = 2). (F and G) CD28 surface expression was monitored by flow cytometry from sorted CD8⁺CD28⁺ during long-term IL-15 treatment (representative, n = 3). (H) SIRT1 protein expression was measured after sorting ex vivo–generated CD28⁺ and CD28⁻ T cells (day 20 of IL-15 treatment); representative blot (n = 2). Data are mean ± SEM of individual donors. **, P < 0.01; ****, P < 0.0001.

Figure 2.

Loss of SIRT1 promotes metabolic reprogramming in resting CD8⁺CD28^– T cells. Metabolism of sorted human CD8⁺ T cell populations was assessed using an extracellular flux (XF) analyzer. (A and B) OCR and ECAR measured in freshly isolated T cell subsets (shown is the mean of biological replicates, n = 7). (C) Glycolytic capacity of T cell subsets (n = 7, paired one-way ANOVA). (D) Energy profile (OCR vs. ECAR) of sorted T cells. Dotted lines indicate corresponding OCR-ECAR data points (shown is the mean of biological replicates, n = 7). (E) ECAR measurement after 48 h of glucose deprivation (representative, n = 2). (F) Lactate concentrations in sorted T cell populations were analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF) after oligomycin treatment (n = 2). (G) Glycolytic capacity of activated and resting T cells (n = 2 biological and 6 technical replicates). (H) ECAR of CD8⁺CD28^– T cells treated with 50 µM RSV for 48 h (shown is the mean of biological replicates, n = 7). (I) Glycolytic capacity of RSV-treated CD8⁺CD28^– T cells (n = 6, paired two-tailed Student’s t test). (J) Energy profile (OCR vs. ECAR) of RSV-treated CD8⁺CD28^– T cells (shown is the mean of biological replicates, n = 7). (K) ECAR of CD8⁺CD28^– T cells treated with nicotinamide riboside for 48 h (representative normalized to 100% baseline, n = 2). Data are mean ± SEM of individual donors. *, P < 0.05; ***, P < 0.001. AA/Rot, antimycin A/rotenone; mpH/min, milli-pH units per minute.

Figure 3.

Altered FoxO1 gene expression in CD8⁺CD28^– T cells. (A) Microarray analysis in CD8⁺CD28⁺ and CD28^– T cells, predicting FoxO1 as an altered transcriptional regulator in CD8⁺CD28^– T cells. (B) CCR7, CD62L, IL7R, and KLRG1 mRNA were assessed by qRT-PCR and normalized to RPL13A mRNA from sorted human T cell populations (n = 5, paired one-way ANOVA). (C) Peripheral blood mononuclear cells were stained for CD3, CD8, CD28, and indicated markers as in B and analyzed by flow cytometry (n = 5, paired one-way ANOVA). (D and E) FoxO1 expression in sorted human T cells was measured by Western blot (representative, n = 7, paired two-tailed Student’s t test). (F) FoxO1 mRNA was analyzed by qRT-PCR and normalized to RPL13A (n = 8). (G and H) CD28⁺ and CD28^– T cells were treated with 20 µM MG132 for 6 h, and FoxO1 expression was measured by Western blot (n = 3, unpaired two-tailed Student’s t test, G shows CD28^– T cells). (I and J) SIRT1 knockdown by Cas9–RNP nucleofection and Western blot for SIRT1 and FoxO1 expression (n = 9, two-way ANOVA). (K) Nucleofection of recombinant SIRT1 protein into CD8⁺ T cells and Western blot for SIRT1 and FoxO1 protein 18 h after nucleofection (representative, n = 2). (L) Densitometry of two independent experiments (n = 2). Data are mean ± SEM of individual donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.000. ns, not significant.

Figure 4.

FoxO1 inhibition reprograms CD8⁺ T cell metabolism and induces cytotoxicity. Metabolism of sorted human CD8⁺ T cell populations was assessed using an extracellular flux analyzer. (A) Glycolytic capacity of sorted human T cell populations treated with 100 nM AS1842856 for 48 h (n = 5, paired two-tailed Student’s t test). (B) Energy profile (OCR vs. ECAR) of T cell subsets treated as in A (shown is the mean of biological replicates, n = 5). (C) Gene expression was measured by qRT-PCR, normalized to RPL13A mRNA, and relative to untreated human CD8⁺ T cells with increasing doses of AS1842856 for 48 h (n = 3, paired two-tailed Student’s t test). (D) Surface marker analysis by flow cytometry after 72 h of incubation with 100 nM AS1842856 (n = 5, two-way ANOVA). (E) Gene expression after CD8⁺ T cell treatment with 100 nM AS1842856 ± glucose for 48 h, measured by qRT-PCR, and normalized to RPL13A mRNA and to untreated cells (n = 5, paired two-tailed Student’s t test). Data are mean ± SEM of individual donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; mpH/min, milli-pH units per minute. (F) Model: a dynamic SIRT1–FoxO1 axis regulates metabolism and cytotoxicity in CD8⁺ T cells. This axis is lost in CD8⁺CD28^– T cells, resulting in increased T cell glycolytic capacity and cytotoxicity.