Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development

Abstract

CD8(+) T cells undergo major metabolic changes upon activation, but how metabolism influences the establishment of long-lived memory T cells after infection remains a key question. We have shown here that CD8(+) memory T cells, but not CD8(+) T effector (Teff) cells, possessed substantial mitochondrial spare respiratory capacity (SRC). SRC is the extra capacity available in cells to produce energy in response to increased stress or work and as such is associated with cellular survival. We found that interleukin-15 (IL-15), a cytokine critical for CD8(+) memory T cells, regulated SRC and oxidative metabolism by promoting mitochondrial biogenesis and expression of carnitine palmitoyl transferase (CPT1a), a metabolic enzyme that controls the rate-limiting step to mitochondrial fatty acid oxidation (FAO). These results show how cytokines control the bioenergetic stability of memory T cells after infection by regulating mitochondrial metabolism.

Figure 1. CD8⁺ T_M cells have substantial mitochondrial spare respiratory capacity

Spleens and lymph nodes were harvested from naive and LmOVA infected mice, and T_N, T_E, and T_M cells were isolated. (A) O₂ consumption rates (OCR) were measured in real time under basal conditions and in response to indicated mitochondrial inhibitors; P < 0.0001 after FCCP injection. Data are representative of 4 independent experiments. (B) Extracellular acidification rates (ECAR) were measured under basal conditions; *P < 0.01 for T_E versus T_N cells, and < 0.01 for T_E versus T_M cells. Data are representative of 2 independent experiments. (C-F) OT-I cells were activated with OVA peptide for 3 days, and subsequently cultured in either IL-2 or IL-15 to generate IL-2 T_E and IL-15 T_M cells, respectively. Basal extracellular acidification rate (ECAR) (D), basal OCR/ECAR ratio (E), and OCR under basal conditions and in response to indicated mitochondrial inhibitors (F) in IL-2 T_E and IL-15 T_M cells are shown; *P < 0.0001 (D), < 0.0001 (E), <0.0001 (F after FCCP). Data are representative of at least 3 independent experiments. Data are shown as mean ± SEM. See also Figure S1.

Figure 2. IL-15 signals promote CD8⁺ T cell survival

OT-I cells were activated with OVA peptide for 3 days, and subsequently cultured in either IL-2 or IL-15. (A) IL-2 T_E and IL-15 T_M cells were re-stimulated with anti-CD3 and anti-CD28 for 5 hours and the amounts of re-stimulation induced ATP are shown relative to non-re-stimulated cells (dashed line); *P = 0.03. Data are shown as mean ± SEM and are representative of 2 independent experiments. (B-C) IL-2 cultured cells were withdrawn from IL-2 for 6–8 hours, and (B) relative Bcl-2 mRNA expression and (C) percentage live cells (based on 7-AAD analysis) are shown. Data are shown as the mean ± SEM (figure generated from 2 independent experiments) (B) or as a representative experiment of 3 independent experiments (C). (D) IL-2 T_E and IL-15 T_M cells were adoptively transferred into congenic recipients (n = 5 per group), and organs were harvested 2 days later, dot plots show percentages, top panels, or total numbers, bottom panels, of donor cells. Data are shown as mean ± SEM. Data are representative of 2 independent experiments. *P = 0.004 (top left), < 0.0001 (top right), 0.005 (bottom left), and < 0.0001 (bottom right). See also Figure S2.

Figure 3. IL-15 induces mitochondrial biogenesis in CD8 ⁺T cells

IL-2 T_E and IL-15 T_M cells were analyzed (A-F). (A) Confocal images show cells stained with Mitotracker (green), Hoechst (blue), and anti-CD8 (white), scale bars are 5 microns. (B) mtDNA/nDNA ratio (figure generated from 2 independent experiments) *P = 0.01, (C) Western analysis for complex I expression, and (D) relative mitochondrial transcription factor A (TFAM) mRNA expression (figure generated from 5 independent experiments) in IL-2 T_E and IL-15 T_M cells; *P = 0.008. (E) Relative total amount of NAD(H), NAD/NADH ratio, and (F) NADH consumed by the ETC (measured as NADH built up after ETC blockade) in IL-2 T_E and IL-15 T_M cells (figures generated from 3 independent experiments); *P = 0.03. Data are shown as mean ± SEM. Data in A and C are representative of at least 3 independent experiments. See also Figure S3.

Figure 4. CD8⁺ T_M cells have greater mitochondrial mass than CD8⁺ T_E cells

Spleens and lymph nodes were harvested from naive and LmOVA infected mice, and T_N, T_E, and T_M cells were isolated. (A) mtDNA/nDNA ratio (data are shown as mean ± SEM, figure generated from 2 independent experiments) *P < 0.01 for T_E vs T_M cells, and < 0.05 for T_N vs T_M cells. (B) Confocal images show T_N, T_E and T_M cells stained with Mitotracker (green) and Hoechst (blue), scale bars are 5 microns. (C) Relative mitochondrial transcription factor A (TFAM) mRNA expression (data are shown as mean ± SEM, figure generated from 2 independent experiments) *P = 0.01, and (D) Western analysis for complex I expression (bars depict quantification of densitometry from 3 blots) *P = 0.008. Data are representative of 2 independent experiments.

Figure 5. Spare respiratory capacity in CD8⁺ T cells is dependent on mitochondrial FAO

(A) Relative CPT1a mRNA expression in IL-2 T_E and IL-15 T_M cells (figure generated from data from 5 independent experiments); *P = 0.003. Oxygen consumption rates (OCR) in IL-15 T_M cells (B), in IL-2 T_E and IL-15 T_M cells (C), and bona fide T_E and T_M cells isolated from LmOVA infected B6 mice (D), under basal conditions and in response to indicated drugs; P < 0.0001 (after FCCP in B left panel), < 0.01 (after etomoxir in B right panel), and < 0.0001 (for T_M cells after etomoxir versus control, D). See also Figure S4.

Figure 6. Spare respiratory capacity in CD8⁺ T cells is dependent on CPT1a expression

Maximum OCR (as indicated by OCR after injection of oligomycin and subsequent FCCP) and spare respiratory capacity (indicated by maximum OCR calculated as percentage of baseline OCR) in IL-15 T_M cells(A, C) and IL-2 T_E cells (B, D) cells transduced with either control (virus expressing shRNA against luciferase) or virus expressing shRNA against CPT1a (hpCPT1a) (A, B), and either control (empty vector) or CPT1a expressing (EX) retrovirus (C, D); *P = 0.03 (A), 0.003 (max OCR) and 0.02 (SRC) (C), 0.006 (max OCR) and 0.03 (SRC) (D). Data are representative of at least 2 independent experiments, and shown as mean ± SEM. See also Figure S5.

Figure 7. Mitochondrial FAO enhances T cell survival and promotes CD8⁺ T_M cell development

OVA peptide activated OT-I cells transduced with control (empty vector) or CPT1a expressing (EX) retrovirus were intravenously injected into congenic recipients (n = 5 per group), and (A) organs were harvested 2 days later, dot plots show percentages (top panels), or total numbers (bottom panels) of donor cells (mean ± SEM); *P < 0.0001 (top left), < 0.0001 (top right), 0.03 (bottom left), and 0.0006 (bottom right), or (B) mice were infected with LmOVA, and peripheral blood CD8⁺ T cells were analyzed by K^bOVA tetramer (line graphs) and GFP for retroviral expression (contour plots) at the peak of the response (T_E cells, day 7) and after contraction (T_M cells, day 14). Contour plots and bar graphs show the frequency of transduced (GFP⁺) cells within the CD8⁺ and K^bOVA tetramer positive gate in blood (left bar graph; *P = 0.0003), absolute numbers of transduced (GFP⁺) cells/ml blood (middle bar graph, data are normalized to control T_E cells; P = 0.02, numbers above bars indicate fold contraction), and absolute numbers of transduced (GFP⁺) cell in spleen (right bar graph, data are normalized to control T_E cells). Data are shown as mean ± SEM and are representative of at least 2 independent experiments with at least 5 mice per group. (C) OT-I cells transduced with control (empty vector) or CPT1a expressing (EX) retrovirus were intravenously injected into IL-15 deficient recipients (n = 5 per group) and mice were infected with LmOVA. Bar graphs show the frequency of transduced (GFP⁺) cells within the K^bOVA tetramer gate at the peak of the response (T_E cells, day 7), after contraction (T_M cells, day 14) and after challenge (recall, day 5 post challenge); *P = 0.01. Data are shown as mean ± SEM and are representative of 2 independent experiments. See also Figure S6.