Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development

Abstract

Generation of CD8(+) memory T cells requires metabolic reprogramming that is characterized by enhanced mitochondrial fatty-acid oxidation (FAO). However, where the fatty acids (FA) that fuel this process come from remains unclear. While CD8(+) memory T cells engage FAO to a greater extent, we found that they acquired substantially fewer long-chain FA from their external environment than CD8(+) effector T (Teff) cells. Rather than using extracellular FA directly, memory T cells used extracellular glucose to support FAO and oxidative phosphorylation (OXPHOS), suggesting that lipids must be synthesized to generate the substrates needed for FAO. We have demonstrated that memory T cells rely on cell intrinsic expression of the lysosomal hydrolase LAL (lysosomal acid lipase) to mobilize FA for FAO and memory T cell development. Our observations link LAL to metabolic reprogramming in lymphocytes and show that cell intrinsic lipolysis is deterministic for memory T cell fate.

Figure 1. Unlike CD8⁺ T_E cells, CD8⁺ T_M cells do not acquire substantial amounts of extracellular fatty acids

OT-I cells were injected i.v. into congenic recipients. Mice were infected with LmOVA to generate T_E (7 days) and T_M (≥ 21 days) cells, and peripheral blood was collected 1h after i.v. Bodipy FL C₁₆ injection and uptake quantified by FACS. (A) Representative plots (histogram) and average MFI (bar graph) of Bodipy FL C₁₆ in T_E and T_M K^bOVA-specific CD8⁺ OT-I⁺ cells from 2 experiments (n=7-8 mice/group). Bar graphs show mean ± SEM, **p <0.01. (B) Representative plots (histogram) of Bodipy FL C₁₆ MFI in polyclonal T_E (CD44^hi CD62L^lo), T_M (CD44^hi CD62L^hi), and T_N (CD44^lo CD62L^hi) CD8⁺ T cells 7 days post-infection and average MFI (bar graph) of polyclonal CD8⁺ T cells from 2 experiments (n=9 mice/group). Bar graphs show mean ± SEM ***p <0.001. (C) OT-I cells were activated with OVA peptide and IL-2 for 3 days and subsequently cultured in IL-15 or IL-2 for 4 more days to generate IL-15 T_M and IL-2 T_E cells respectively. (D) OTI cells were incubated with Bodipy FL C₁₆ on d4-7 of culture, and uptake was measured. MFIs were normalized to the daily MFI of IL-2 T_E cells. Data from 3 experiments shown as mean ± SEM, *p <0.05 by one sample t-test. (E) CD36 expression on d7 IL-2 T_E and IL-15 T_M cells, or polyclonal T cells. Data represent 3 experiments. (F-H) Day 6 IL-2 T_E or IL-15 T_M cells were cultured overnight ± oleate then stained with Bodipy. (F) Representative plots showing Bodipy MFI and (G) average Bodipy MFI from 3 experiments show mean ± SEM, *p <0.05, **p <0.01. (H) Images showing Bodipy (green) and CD8 (red) represent 4 experiments. Scale bar = 2 µM (I) OT-I cells expressing a lipid droplet-targeting construct (LD-GFP) were cultured overnight ± oleate. Images show LD-GFP (green) and CD8 (red) and represent 2 experiments. (J) Cells were incubated with Bodipy-LDL or left unstained (Control), data represent 2 experiments.

Figure 2. Ultrastructure analysis reveals densely packed mitochondria in close proximity to ER in T_M cells

EM images of mitochondria associated with ER from (A) T_E and T_M cells and (B) IL-2 T_E and IL-15 T_M cells; mitochondrion, red arrow; ER, blue arrow; electron dense structure, green arrow. Data represent ≥ 2 experiments.

Figure 3. Glucose fuels mitochondrial FAO and OXPHOS in IL-15 T_M cells

(A) IL-2 T_E and IL-15 T_M cell survival (7-AAD exclusion) ± C75, shown as survival relative to vehicle (Veh.) treated cells. Data from 5 experiments show mean ± SEM, **p <0.01. (B) IL-2 T_E proliferation in the presence of C75 or vehicle. Data represent 3 experiments. (C) OCR of IL-2 T_E or IL-15 T_M cells cultured in TCM (Control) and low glucose TCM (LG) was measured under basal conditions and in response to oligomycin (Oligo), FCCP, etomoxir (Eto) and rotenone + antimycin (Rot+Ant). Data represent 2 experiments. (D) Representative plot (histogram) and average MFI (bar graph) of 2-NBDG uptake in polyclonal T_E and T_M cells from 1 experiment (n=4 mice/group). Data in bar graph show mean ± SEM, ***p <0.001. (E) OCR represented as % of baseline for IL-15 T_M cells cultured with AmA or vehicle (Control) measured in response to indicated drugs. Data represent 2 experiments. (F) OCR of IL-2 T_E or IL-15 T_M cells cultured in TCM (Control) and lipid depleted TCM (LD), measured under basal conditions and in response to indicated drugs. Data represent 2 experiments.

Figure 4. The lipid signature of T_M cells suggests active lipolysis

(A-F) Lipids were quantified from d7 cultured IL-2 T_E and IL-15 T_M cells. Data represent 3 experiments shown as mean ± SEM, *p <0.05.

Figure 5. Lysosomes associate with intracellular neutral lipids during the T_M cell transition in vitro

(A) Relative mRNA expression of LAL in d7 IL-2 T_E and IL-15 T_M cells. Data from 3 experiments shown as mean ± SEM, ***p <0.001. (B) LAL protein expression compared to β-Actin. Data represent 2 experiments. (C) Relative mRNA expression of LAL in T_E and T_M cells. Data from 3 experiments shown as mean ± SEM, *p <0.05. IL-2 T_E and IL-15 T_M cells were stained with LysoTracker (blue) and Bodipy (green) on d4 (D) or d7 (E) of culture. Scale bar = 2 µM. Data represent 3 experiments.

Figure 6. Lysosomal lipolysis metabolically programs CD8⁺ T cells toward the T_M cell phenotype

IL-2 T_E or IL-15 T_M cells were transduced with retroviral vectors containing shRNA against luciferase (Control) or against LAL (hpLAL). (A) Cells were stained for neutral lipids with Bodipy. Data represent 3 experiments. (B) IL-15 T_M cells were analyzed for lipid content by MS. Data from 3 experiments show mean ± SEM, *p <0.05, ***p <0.001. (C-E) On day 3 of culture, IL-2 T_E cells were washed and resuspended in media with IL 15. OCR and ECAR were measured. (C) Representative plot showing OCR. (D) Relative change in OCR, ECAR and OCR/ECAR 5 hours after baseline, represented as % change from baseline. Data from 5 experiments show mean ± SEM, *p <0.05 or **p <0.01. (E) Expression of CD25 and CD62L on d7 IL-2 T_E or IL-15 T_M cells. Data represent 5 experiments. (F) Day 3 IL-2 T_E cells were cultured in IL-2 or IL-15 and analyzed daily (d0-4) for survival (7AAD exclusion). Data represented as % survival relative to non-transduced cells. Data from 3 experiments shown as mean ± SEM, **p <0.01 by 2 way ANOVA.

Figure 7. T cell intrinsic lysosomal lipolysis supports CD8⁺ T_M cell development after infection

(A-B) Congenic IL-2 T_E cells were transduced with control or hpLAL and sorted on GFP. On d6 of culture, 2x10⁶ cells/mouse were injected i.v. and harvested from spleen or LN 2 days later. Data represent 3 experiments, shown as mean ± SEM, ***p <0.001. (C-E) Congenic OT-I control or hpLAL transduced cells were transferred into mice infected 1 day prior with LmOVA. (C-D) Blood was analyzed for CD8, K^bOVA, Thy1.1, and GFP. (C) Data shown as % of total CD8⁺ (line graph); representative plots show % GFP⁺ of Thy 1.1⁺ donor cells. Data represent 2 experiments, *p <0.05. (D) Mice were challenged with LmOVA and 5 days later CD8⁺ K^bOVA GFP⁺ cells from spleen were quantified. Data shown as mean ± SEM and represent 2 experiments, **p <0.01.