CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability

Abstract

A characteristic of memory T (TM) cells is their ability to mount faster and stronger responses to reinfection than naïve T (TN) cells do in response to an initial infection. However, the mechanisms that allow this rapid recall are not completely understood. We found that CD8 TM cells have more mitochondrial mass than CD8 TN cells and, that upon activation, the resulting secondary effector T (TE) cells proliferate more quickly, produce more cytokines, and maintain greater ATP levels than primary effector T cells. We also found that after activation, TM cells increase oxidative phosphorylation and aerobic glycolysis and sustain this increase to a greater extent than TN cells, suggesting that greater mitochondrial mass in TM cells not only promotes oxidative capacity, but also glycolytic capacity. We show that mitochondrial ATP is essential for the rapid induction of glycolysis in response to activation and the initiation of proliferation of both TN and TM cells. We also found that fatty acid oxidation is needed for TM cells to rapidly respond upon restimulation. Finally, we show that dissociation of the glycolysis enzyme hexokinase from mitochondria impairs proliferation and blocks the rapid induction of glycolysis upon T-cell receptor stimulation in TM cells. Our results demonstrate that greater mitochondrial mass endows TM cells with a bioenergetic advantage that underlies their ability to rapidly recall in response to reinfection.

Keywords: lymphocytes; metabolism.

Fig. 1.

IL-15 T_M cells have more mitochondrial mass than T_N cells. OT-I cells were activated with OVA peptide for 3 d in IL-2 and cultured in IL-15 for 3–4 d to generate IL-15 T_M cells, which were compared with OT-I T_N cells. (A) T_N and IL-15 T_M cells stained with Mitotracker green (green) and DRAQ5 (blue). (B) Mitotracker green staining was quantified by flow cytometry. Data are representative of three experiments. (C) mtDNA/nDNA ratio, mean ± SEM, from six experiments; *P < 0.05.

Fig. 2.

Secondary T_E cells proliferate faster and make more IFN-γ than primary T_E cells. Naïve OT-I and IL-15 T_M cells were (re)stimulated with anti-CD3/28 for 3 d. (A) Proliferation by Cell Trace Violet dilution at indicated time points. Bar graphs represent difference in mean fluorescence intensity (MFI) at the indicated time point relative to t = 0. (B) IFN-γ production 2 d after (re)stimulation; representative of ≥2 experiments.

Fig. 3.

Enhanced secondary T_E cell proliferation is marked by greater metabolic activity and ATP production. Naïve OT-I and IL-15 T_M cells were (re)stimulated with anti-CD3/28. (A) Proliferation and Mitotracker deep red staining 1 and 2 d after (re)stimulation. (B) OCR and ECAR in primary and secondary T_E cells 2 d after stimulation; mean ± SEM; *P < 0.0001. (C and D) ATP in primary and secondary T_E cells 3 and 24 h after (re)stimulation with anti-CD3/28 (C), and in naïve and IL-15 T_M cells (D). Data are shown as mean ± SEM; *P < 0.005 (C) and < 0.001 (D); representative of ≥2 experiments.

Fig. 4.

IL-15 T_M cells have enhanced glycolytic capacity compared with T_N cells after activation. (A) Basal ECAR and OCR were measured in OT-I T_N and IL-15 T_M cells; *P < 0.0001. T_N and IL-15 T_M cells were stimulated with anti-CD3/28 beads (B) or with PMA/iono (C) and OCR and ECAR measured. Data in A–C are from the same experiment and are representative of ≥3 experiments. (D) Compiled and baselined data as shown in C, from two experiments; peak is first measurement after PMA/iono, plateau is at 120 min. *P < 0.0001 (Left), and < 0.05 and < 0.001 (Right); mean ± SEM.

Fig. 5.

Mitochondria-derived ATP is required for the initiation of proliferation and facilitates glycolysis in IL-15 T_M cells after PMA/iono. (A) OT-I T_N and IL-15 T_M cells were (re)stimulated with anti-CD3/28 with or without oligo (added at day 0), and proliferation is shown at days 0 and 2; representative of four experiments. (B) OT-I cells were activated with OVA peptide and IL-2 for 3 d, then labeled with Cell Trace Violet and oligo added (day 0). Proliferation was observed at days 0, 2, and 3; representative of two experiments. (C) Relative OCR in primary and secondary T_E cells 2 d after anti-CD3/28, ±5 nM oligo; mean ± SEM, representative of two experiments; *P < 0.0001. (D) IL-15 T_M cells were restimulated with PMA/iono, with or without oligo, and OCR and ECAR were measured. Data are shown as mean ± SEM, representative of three experiments.

Fig. 6.

T_M cells have enhanced glycolysis, more ATP, and proliferate faster than T_N cells after activation. CD8 T_N (CD44^lo CD62L^hi) and T_M (CD44^hi CD62L^hi) cells were isolated from naïve and LmOVA-infected mice. OCR and ECAR in T_N and T_M cells after stimulation with aCD3/28 coated beads (A), and after subsequent oligo (1 μM), FCCP (1.5 μM), and rotenone (100 nM) plus antimycin A (1 µM) injections (B) (data in A and B are from the same experiment). (C) T_N and T_M cells were PMA/iono stimulated, exposed to oligo + FCCP, and R+A, and OCR and ECAR measured. T_N is the same as Fig. S4, T_M is the same as Fig. 8F and Fig. S7. (D) Compiled and baselined data as shown in C, from two experiments; peak is first measurement after PMA/iono, plateau is at 120 min. *P < 0.0005 (Left), and < 0.0001 (Right). Forward scatter and ATP of resting T_N and T_M cells (E), and ATP in primary and secondary T_E cells 3 and 24 h after aCD3/28 (F). (G) T_N and T_M cells stimulated with aCD3/28 with or without oligo (day 0) and proliferation is shown. T_N control is the same as in Figs. S6B and S12B. T_M control is the same as Fig. 7A and 8E; mean ± SEM, representative of two (A, B, and E), 3 (C), or one (F and G) experiment(s).

Fig. 7.

FAO fuels the OXPHOS needed for rapid recall of T_M cells. (A) Proliferation of T_M cells from LmOVA-infected mice after anti-CD3/28 ± etomoxir (day 0); control is same as Fig. 6G and 8E. Data are from one experiment. (B) Proliferation of IL-15 T_M cells after anti-CD3/28 ± etomoxir; representative of four experiments. (C) OCR and ECAR of IL-15 T_M cells after PMA/iono ± etomoxir; mean ± SEM, representative of four experiments. (D–F) Proliferation of IL-15 T_M cells expressing control (shRNA against luciferase), hpCPT1a (shRNA against CPT1a), or hpMPC1 (shRNA against MPC1) retrovirus after anti-CD3/28 (D); graph shows percent of cells normalized to control cells in gate with fewer divisions (separated by line), generated from two experiments. (E) OCR and ECAR of control, hpCPT1a, and hpMPC1 IL-15 T_M cells after PMA/iono. (F) Compiled and baselined data as shown in E, from three experiments; peak is at first measurement after PMA/iono, plateau is at 120 min. n.s., not significant. *P < 0.05; mean ± SEM (E and F) and representative of two (D) or four (E) experiments.

Fig. 8.

Dissociation of HK from mitochondria impairs proliferation and the rapid engagement of glycolysis in (IL-15) T_M cells after activation. (A) Western blot analysis for HK I and II in IL-15 T_M cells incubated ± CLT for 2 h; GAPDH and prohibitin I are loading controls for cytosolic and mitochondrial fractions, respectively; representative of two experiments. (B) OCR and ECAR of IL-15 T_M cells stimulated with PMA/iono in the presence of control or HK-1 peptide (10 μM) or CLT (25 μM); mean ± SEM, representative of ≥2 experiments. IL-15 T_M cells were stimulated with anti-CD3/28 and proliferation ± CLT; representative of five experiments (C), or with control or HK-1 peptide (10 μM); representative of two experiments, is shown (D). (E and F) T_M cells from LmOVA-infected mice. Proliferation after anti-CD3/28 ± CLT; control is same as Fig. 6G and 7A, from one experiment (E); OCR and ECAR after PMA/iono ± CLT and exposed to oligo (1 μM) plus FCCP (1.5 μM), and rotenone (100 nM) plus antimycin A (1 µM); mean ± SEM and representative of two experiments; control is the same as Fig. 6C and Fig. S7 (F).