Metabolic reprogramming in memory CD4 T cell responses of old adults

Abstract

To determine whether aging affects the ability of T cells to undergo metabolic reprogramming upon activation, we compared CD4 T cell responses after polyclonal in vitro stimulation. Compared to younger adults, CD4 memory T cells from healthy older individuals exhibited a higher upregulation of oxidative phosphorylation with increased production of reactive oxygen species and intracellular and secreted ATP. Increased ATP secretion led to increased purinergic signaling and P2X7-dependent increases in cytoplasmic calcium. The increased mitochondrial activity was not due to a difference in activation-induced mitochondrial biogenesis. Expression of carnitine palmitoyl transferase 1 was higher, conversely that of fatty acid synthase was reduced in older T cells, resulting in increased fatty acid oxidation, while depleting intracellular lipid stores. The aged CD4 memory T cells therefore maintain a more catabolic state in lipid metabolism, while their ability to upregulate glycolysis upon activation is preserved.

Keywords: Aging; CD4 memory T cells; Fatty acid oxidation; Immunometabolism; Immunosenescence; T cell activation.

Figure 1.. Increased activation-induced mitochondrial respiration in CD4 memory T cells with age.

(A) MitoTracker staining at indicated time points after Dynabead activation of memory CD4 T cells are shown. Results from day 0 to day 2 are shown as mean±SEM from 10 to 11 adults; day 3 is mean±SEM from 5 adults. (B and C) Memory CD4 T cells from healthy adults were activated with anti-CD3/CD28 Dynabeads. Basal O₂ consumption rates (OCR, B) and extracellular acidification rates (ECAR, C) were determined at indicated time points using Seahorse Analyzer. Kinetic data at 0h (n=3), 12h (n=5), 24h (n=8), 48h (n=14); 72h (n=11); 96h (n=4) after stimulation are shown as mean±SEM. (D) Mean±SEM ECAR from day 2 activated memory CD4 T cells from young and old individuals. Statistical analysis by unpaired two-tailed t-test. E) Real-time OCR from CD4 memory T cells two days after activation are shown under basal conditions and in response to indicated mitochondrial inhibitors. Mean±SEM of triplicate measurement of cells from one young and one old individual run in parallel. (F-H) Mean±SEM basal OCR (F), ATP-coupled OCR (G) and SRC (H) of samples in (D).

Figure 2.. Increased activation-induced mitochondrial ROS and ATP production by CD4 memory T cells with age

(A) MFI of MitoSox indicative of mitochondrial ROS in CD4 T cells from 6 young and 8 old adults two days after activation. Statistical analysis by unpaired two-tailed t-test. (B) Intracellular ATP in day 2-activated memory CD4 T cells (n=9 pairs). (C) Extracellular levels of ATP from supernatants of activated young and old memory CD4 T cells (n=3 pairs). Comparisons were done by unpaired one-tailed t-test. (D) Cultures with CD4 memory T cells were performed in the presence of vehicle or the P2X7 receptor inhibitors. Intracellular calcium concentrations were determined by Fluo8 staining and cytometric analysis of memory CD4 T cells two days after activation. (E) Calcium staining is performed as described in (D) and results from 10 young and 10 older adults, always run in pairs are shown (right). One representative flow cytometry staining graph of one young and one old is shown on left.

Figure 3.. Metabolic pathways driving respiratory activity in activated CD4 memory T cells with age.

(A) MitoTracker staining of two-day activated CD4 memory T cells from 4 young and 6 older adults. (B) Western blot of cytochrome C expression by memory CD4 T cells from 9 young and 9 older adults on day 2 after activation. Comparisons were done by two-tailed unpaired t-test. (C, D and E) Real-time OCR from CD4 memory activated for 2 days. UK5099 to inhibit pyruvate transport into the mitochondria was added as indicated (C). Glutaminolysis was inhibited by adding BPTES (D). Fatty acid oxidation was inhibited by adding Etomoxir (E). Results are shown as mean±SEM of triplicate cultures. (F) Summary for FAO-, pyruvate-, and glutamine-coupled mitochondrial OCR as shown in (C, D and E) from 3, 2 and 3 pairs of young and old individuals, respectively. Comparison by unpaired two-tailed t-test. (G) Representative images of BODIPY staining of activated CD4 memory T cells from one young and one old individual (left) and summary from three experiments are shown (right). Scale bars, 10 μm.

Figure 4.. Decreased FAS and increased CPT1a expression in activated CD4 memory T cells with age.

(A) Representative images of FAS staining in activated CD4 memory T cells from one young and one old individual (left) and summary data from three experiments are shown (right). Scale bars, 10 μm. (B) CPT1a expression levels were determined by Peggy Sue western. A representative blot of signal intensities from activated CD4 memory T cells from a young and an old adult (left) and the ratio of CPT1a to actin from 16 pairs (right) are shown. Comparisons by unpaired one-tailed t-test. (C) Memory CD4 T cells were transfected with siRNA for CPT1a and activated for two days. Knockdown efficiency by Peggy Sue western (left) and basal OCR in five control and siCPT1a-silenced cells (right) are shown. Comparisons were done by paired one-tailed t-test. (D) Extracellular ATP concentrations in supernatants from activated CD4 memory T cells transfected with control or CPT1a-siRNA (n=3). Comparison by one-tailed paired t-test.

Figure 5.. Increased SIRT1 expression in old memory CD4 T cells leads to increased CPT1a expression.

(A) AMPK phosphorylation was compared by Peggy Sue western in day 2 activated CD4 memory T cells from 12 young and 12 older adults. Representative blot of signal intensities (left) and summary data (right). Comparison by unpaired one-tailed t-test. (B) Activated CD4 T memory cells were transfected with siRNA for AMPKα on day 1. Knockdown efficiency by qPCR (left) and CPT1a expression by Peggy Sue western (right) are shown on day 2 after activation (n=9). (C) SIRT1 expression in day 2 activated CD4 memory T cells were determined by Peggy Sue western. A representative blot of signal intensities from a young and an old adult (left) and the ratio of SIRT1 to actin from the samples shown in (A) are shown. (D) Activated CD4 T memory cells were transfected with siRNA for SIRT1 on day 1. Knockdown efficiency by Peggy Sue western (left) and CPT1a expression by qPCR (middle) and Peggy Sue western (right) are shown on day 2 after activation (n=6). Comparisons were done by paired one-tailed t-test.