Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species

Abstract

The AMP-activated kinase (AMPK) is a major energy sensor metabolic enzyme that is activated early during T cell immune responses but its role in the generation of effector T cells is still controversial. Using both in vitro and in vivo models of T cell proliferation, we show herein that AMPK is dispensable for early TCR signaling and short-term proliferation but required for sustained long-term T cell proliferation and effector/memory T cell survival. In particular, AMPK promoted accumulation of effector/memory T cells in competitive homeostatic proliferation settings. Transplantation of AMPK-deficient hematopoïetic cells into allogeneic host recipients led to a reduced graft-versus-host disease, further bolstering a role for AMPK in the expansion and pathogenicity of effector T cells. Mechanistically, AMPK expression enhances the mitochondrial membrane potential of T cells, limits reactive oxygen species (ROS) production, and resolves ROS-mediated toxicity. Moreover, dampening ROS production alleviates the proliferative defect of AMPK-deficient T cells, therefore indicating a role for an AMPK-mediated ROS control of T cell fitness.

Figure 1

AMPK promotes peripheral T cells replenishment upon bone marrow transfer. (A, B) Relative contribution of AMPK-KO CD4⁺ (A) and CD8⁺ (B) T cells in the thymus, spleen, MLN, LP and IEL of CD3ε^−/− mice reconstituted with a 1:1 mixture of bone marrow cells from WT (CD45.1) and AMPK^KO-T (CD45.2) mice. Data are expressed as a ratio of AMPK-KO over WT cells in the CD4⁺ or CD8⁺ T cell gate. (C–H) CD62L, CD44 and CXCR3 expression by WT and AMPK-KO T cells recovered from the spleen of mice reconstituted as in (A, B). Representative dot plots (C, E) and histograms showing the mean % ± SD (D, F) in different cell gates as indicated. Data are representative of 3 (A, C, D, G, H) or 2 (B, E, F) independent experiments with n = 6. Statistical analysis: Friedman followed by Dunn multiple comparison (A, B), Mann–Whitney (D, F), Wilcoxon Test (H). *p < 0.05 ; **p < 0.01 ; ***p < 0.001. MLN mesenteric lymph nodes, LP lamina propria, IEL intra-epithelial lymphocytes.

Figure 2

WT CD4⁺ T cells outcompete their AMPK-KO counterparts during homeostatic proliferation. CFSE labeled CD4⁺ naive T cells from WT (CD45.1) and AMPK^KO-T (CD45.2) mice (1:1 ratio) were i.v. injected into CD3ε^−/− mice. Recovered T cells were analyzed on day 10 by flow cytometry. (A, B) Relative contribution of AMPK-KO T cells to the repopulation of the CD4⁺ T cell subset in the spleen, MLN, LP and IEL (CD4⁺ gated). Representative dot plots (A) and histograms showing the ratio of AMPK-KO/WT cells (B, gate CD4⁺ T cells). Data are representative of 3 (LP, IEL), 4 (MLN) or 6 (spleen) independent experiments with n = 6. (C) CFSE dilution versus CD45.1 expression by CD4⁺ T cells in the spleen. (D) Relative contribution of AMPK-KO T cells in the pool of recovered splenic CD4⁺ T cells that divided 0–2 or > 3 times, according to CFSE dilution (pool of 4 independent experiments with n = 15). (E) Percentage of WT and AMPK-KO CD4⁺ T cells that express Ki67 in the spleen (1 experiment representative of 2, with n = 5). (F–H) Percentage of WT and AMPK-KO CD4⁺ T cells expressing CXCR3 (F) or producing IL-2 and IFNγ (H) according to the number of cell divisions in the spleen. (G–J) Percentage of WT and AMPK-KO CD4⁺ T cells expressing CXCR3 (G), IL-2 (I) or IFNγ (J) among CD4⁺ T cells that divided more than 3 times (pool of 2–3 independent experiments, with n = 7–10). Statistical analysis: Friedman followed by Dunn multiple comparison (B), Wilcoxon (D, E, G, I and J). *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 3

AMPK promotes homeostatic proliferation in vitro. (A) Naive Th cells from C57BL/6 mice were cultured in the presence of IL-7 and/or syngeneic DCs, as indicated. Culture media were replaced with fresh DCs and IL-7 each 4–5 days. Results indicate the numbers of T cells recovered on day 18, with dashed line corresponding to the number of cells in the input culture (day 0) (data are pooled from 2 independent experiments, n = 3–6). (B) Relative recovery of AMPK-KO T cells after 14 day-culture with IL-7 in the presence or absence of syngenic DC. (C) Relative recovery of AMPK-KO T cells at day 6 and 14 of IL-7 + DC culture. (D) CFSE dilution profiles of WT and AMPK-KO at day 14 of IL-7 + DC culture. (E) Relative contribution of AMPK-KO T cells according to CFSE content. (F) Percentage of WT and AMPK-KO T cells expressing IFNγ at day 14. Data are representative of at least 5 experiments with n = 17 (B) or 8 (C), or are pool of 2 independent experiments with n = 3–6 (A), 13 (E) or 11 (F). Statistical analysis: Mann–Whitney (B), Friedman followed by Dunn multiple comparison (C) Wilcoxon (E–F). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

AMPK promotes mitochondrial fitness of T cells. (A–C) Mitochondrial membrane potential measured as a ratio of JC1 Red/Green fluorescence ratio in naive T cells (A), homeostatic proliferation-recovered T cells (B) and memory-like Th1 cells (C), with WT ratio set to 1. (D–F) Basal OCR (D), maximal OCR (E) and ECAR (F) of WT and AMPK-KO T cells recovered after 14 days of homeostatic proliferation. (G–I) TEM analysis of WT and AMPK-KO naive T cells. Representative images (G), numbers (H) and areas (I) of mitochondria (a total of 179 (WT) and 170 (KO) mitochondria from 27 (WT) and 29 (AMPK-KO) T cells analyzed are represented). (J, K) Image representative (J) and percentage (K) of mitochondria presenting a budding protrusion (WT : 3 out of 179, AMPK-KO : 16 out 170 mitochondria). Data are representative of 7 (A, B) or 2 (H, I, K) experiments or are pooled from 9 (C) or 2 (D–F, n = 6) independent experiments. Statistical analysis: paired t-test (A–C), Mann–Whitney (D–F, H–I), Chi² (K). **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Accelerated mitochondrial clearance and defective mitochondrial stress-induced glycolytic switch in AMPK-KO T cells. (A) Representative FACS profiles of total mitochondrial mass (left panel) and pulse-chase remaining mitochondrial staining (right panels) in WT and AMPK-KO Th cells after 4, 6 and 9 day in IL-7-supplemented media. (B) Kinetic of mitochondrial mass loss, calculated from gate indicated in (A, right panels). (C, D) Kinetic of ECAR upon oligomycin treatment of WT and AMPK-KO Th1 memory-like cells (C) or IL-7 + DC-expanded T cells (D). Data are expressed as fold increase relative to time point 0. Raw data are also provided in supplemental Figure S13. Data are pooled from 3 independent experiments (n = 9–16, B) or 2 independent experiments (n = 6, D), or are representative of 7 individual experiments (n = 16, C). Statistical analysis: Wilcoxon (B), Mann-Withney (C, D). **p < 0.01; ****p < 0.0001.

Figure 6

AMPK dampens ROS production and toxicity in T cells. (A–C) Cellular ROS staining with DHR123 probe in IL-7 + DC-expanded T cells (A), in memory-like Th1 cells (B), and in naive T cells (C). FACS staining of a representative experiments (left panels) and histogram showing MFI fold increase in AMPK-KO cells in comparison to WT cells set to 1 (right panels). (D, E) Percentages of cell viability after treatement of WT and AMPK-KO naive Th cells with H₂O₂ (0.5 mM for 2 h, D) or with staurosporine (STS, 5 µM for 4 h, E). (F, H) Cellular ROS staining of naive Th cells after 4 days in IL-7-supplemented culture medium with or without 2-ME (F) and supplemented with MitoTEMPO (H). (G, I) Relative recovery of AMPK-KO T cells after 14 days of homeostatic proliferation in vitro in a culture medium with or without 2-ME (G) and with or without MitoTEMPO (I). Data are pooled from 7 (n = 11, A), 9 (n = 8, B), (n = 7, C), 3 (n = 5, D), 4 (n = 4, E), 4 (n = 20, G) or 6 (n = 13, I) independent experiments or are representative of 3 individal experiments (F, H). Statistical analysis: Wilcoxon (A-C), Mann–Whitney (D, E, G, I). *p < 0.05; **p < 0.01; ****p < 0.0001.