Mitochondrial Dynamics Controls T Cell Fate through Metabolic Programming

Abstract

Activated effector T (TE) cells augment anabolic pathways of metabolism, such as aerobic glycolysis, while memory T (TM) cells engage catabolic pathways, like fatty acid oxidation (FAO). However, signals that drive these differences remain unclear. Mitochondria are metabolic organelles that actively transform their ultrastructure. Therefore, we questioned whether mitochondrial dynamics controls T cell metabolism. We show that TE cells have punctate mitochondria, while TM cells maintain fused networks. The fusion protein Opa1 is required for TM, but not TE cells after infection, and enforcing fusion in TE cells imposes TM cell characteristics and enhances antitumor function. Our data suggest that, by altering cristae morphology, fusion in TM cells configures electron transport chain (ETC) complex associations favoring oxidative phosphorylation (OXPHOS) and FAO, while fission in TE cells leads to cristae expansion, reducing ETC efficiency and promoting aerobic glycolysis. Thus, mitochondrial remodeling is a signaling mechanism that instructs T cell metabolic programming.

Figure 1. Effector and memory T cells possess distinct mitochondrial morphologies

(A) Effector (T_E, CD44^hi CD62L^lo, 7 days post infection) and memory T (T_M, CD44^hi CD62L^hi, 21 days post infection) cells sorted from C57BL/6 mice infected i.p. with 10⁷ CFU LmOVA and (B) IL-2 T_E and IL-15 T_M cells generated from differential culture of OT-I cells activated with OVA peptide and IL-2 using IL-2 or IL-15 analyzed by EM, scale bar = 0.5 µm. (C–D) Mitochondrial morphology in live OT-I PhAM cells before and after αCD3/CD28 activation and differential cytokine culture by spinning disk confocal microscopy. Mitochondria are green (GFP) and nuclei are blue (Hoechst), (C) scale bar = 5 µm, (D) scale bar = 1 µm. (E) Immunoblot analysis of cell protein extracts from (C), probed for Mfn2, Opa1, Drp1, phosphorylated Drp1 at Ser616 (Drp1^pS616), and β-actin. (A–E) Representative of 2 experiments. See also Figure S1.

Figure 2. Memory T cell development and survival, unlike effectors, requires mitochondrial fusion

(A) Relative in vitro survival ratios of Mfn1, Mfn2, or Opa1 deficient (CD4 Cre⁺, −/−) to wild-type (CD4 Cre^-, +/+) OT-I IL-2 T_E and IL-15 T_M cells (*p=0.0465). Data normalized from 2–3 independent experiments shown as mean ± SEM. (B) Mitochondrial morphology of OT-I Opa1 wild-type and Opa1^−/− IL-2 T_E and IL-15 T_M cells analyzed by EM (scale bar = 0.5 µm, represents one experiment) and (C) Seahorse EFA. (Left) bar graph represents ratios of O₂ consumption rates (OCR, indicator of OXPHOS) to extracellular acidification rates (ECAR, indicator of aerobic glycolysis) at baseline and (right) spare respiratory capacity (SRC) (% max OCR after FCCP injection of baseline OCR) of indicated cells (*p<0.03, **p=0.0079). Data from 3 experiments shown as mean ± SEM. (D–F) 10⁴ OT-I Opa1^+/+ or Opa1^−/− T cells were transferred i.v. into C57BL/6 CD90.1 mice infected i.v. with 10⁷ CFU LmOVA. Blood analyzed by flow cytometry at indicated times post infection. After 21 days, mice were challenged i.v. with 5×10⁷ CFU LmOVA and blood analyzed post challenge (p.c.). (D) % Donor K^b/OVA⁺ CD90.2⁺ cells shown in representative flow plots and (E) line graph with mean ± SEM (*p=0.0238, **p<0.005). (F) Number of donor K^b/OVA⁺ cells from spleens of infected mice shown with mean ± SEM (*p=0.0126). (D–F) Representative of 2 experiments (n=9–11/ group). See also Figure S2.

Figure 3. Enhancing mitochondrial fusion promotes the generation of memory-like T cells

(A–F, I–L) OVA peptide and IL-2 activated OT-I cells differentiated into IL-2 T_E or IL-15 T_M cells for 3 days in the presence of DMSO or 20 µM fusion promoter M1 and 10 µM fission inhibitor Mdivi-1 (M1+Mdivi-1) as shown (A) pictorially. (B) Representative spinning disk confocal images from 3 experiments of live cells from OT-I PhAM mice. Mitochondria are green (GFP) and nuclei are blue (Hoechst), scale bar = 5 µm. (C) Cells stained with MitoTracker Green and analyzed by flow cytometry. Relative MFI (left) from 6 experiments (*p=0.0394, **p=0.0019) with representative histograms (right). (D) Baseline OCR and SRC from 3–4 experiments (*p=0.0485, ***p<0.0001) and (E) CD62L expression analyzed by flow cytometry of indicated cells. Relative MFI (left) from 7 experiments (*p=0.0325, **p=0.0019, ***p<0.0001) with representative histograms (right). (F) OCR of indicated cells at baseline and in response to PMA and ionomycin stimulation (PMA+iono), oligomycin (Oligo), FCCP, and rotenone plus antimycin A (R+A). Represents 2 experiments. (C–F) Shown as mean ± SEM. (G–H) OT-I cells were transduced with either empty (Control), Mfn1, Mfn2, or Opa1 expression vectors, sorted, and cultured to generate IL-2 T_E cells. (G) Representative histograms of MitoTracker Deep Red staining from 4 experiments and (H) basal OCR from 2 experiments of transduced cells. (I–L) 1–2×10⁶ IL-2 T_E cells cultured with DMSO (gray diamonds) or M1+Mdivi-1 (blue squares) were transferred into congenic C57BL/6 recipient mice. Cell counts of donor cells recovered 2 days later from the (I) spleen (***p=0.005) and (J) peripheral lymph nodes (pLNs, ***p=0.0006). Dots are individual mice. (K) Blood from recipient mice analyzed for % donor K^b/OVA⁺ cells post transfer and challenge with 10⁷ CFU LmOVA by flow cytometry (*p=0.0150, n=5/group). (L) Donor K^b/OVA⁺ cells recovered from recipient spleens 6 days p.c. (*p=0.0383). Dots are individual mice (I–L) Represents 2 experiments shown with mean ± SEM. See also Figure S3.

Figure 4. Mitochondrial fusion improves adoptive cellular immunotherapy against tumors

(A–B) C57BL/6 mice inoculated s.c. with 10⁶ EL4-OVA cells. (A) After 5 or (B) 12 days, 10⁶ or 5×10⁶ OT-I IL-2 T_E cells cultured with DMSO or M1+Mdivi-1 were transferred i.v. into recipients and tumor growth assessed. Represents 2 experiments shown as mean ± SEM (n=5/group, *p<0.05, **p<0.005). (C-E) Human CD8⁺ PBMCs activated with αCD3/CD28 + IL-2 to generate IL-2 T_E cells. (C) Confocal images of indicated cells where mitochondria are green (MitoTracker) and nuclei are blue (Hoechst). Representative images from 2 of 4 biological donors, scale bar = 5 µm. (D) OCR/ECAR ratios and SRC of indicated cells from 2 separate donors shown as mean ± SEM (*p=0.0303, **p<0.005, ***p<0.0001). (E) MitoTracker Green staining and CD62L, CD45RO, and CCR7 expression analyzed by flow cytometry shown with representative histograms from 4–6 biological replicates. See also Figure S4.

Figure 5. Fusion promotes memory T cell metabolism, but Opa1 is not required for FAO

(A–E) OCR measured at baseline and in response to media, etomoxir (Eto) and other drugs as indicated of (A) IL-2 T_E cells cultured in DMSO or M1+Mdivi-1, (B) control or Opa1 transduced IL-2 T_E cells, (C) Opa1^+/+ and Opa1^−/− IL-2 T_E cells cultured in DMSO or M1+Mdivi-1 (D) or without drugs, and (E) ex vivo donor OT-I Opa1^+/+ and Opa1^−/− day 7 T_E cells derived from LmOVA infection. (A-E) Representative of 2 experiments shown as mean ± SEM (***p<0.0001). See also Figure S5.

Figure 6. Mitochondrial cristae remodeling signals metabolic pathway engagement

(A) Basal ECAR of OT-I Opa1^+/+ and Opa1^−/− IL-2 T_E cells (left) and day 7 T_E cells isolated ex vivo after adoptive transfer from LmOVA infection (right). Data combined from 2–3 experiments (*p=0.0412, ***p<0.0001). (B) OCR at baseline and with indicated drugs, representative of 2 experiments shown as mean ± SEM and (C) D-Glucose-¹³C_1,2 trace analysis of OT-I Opa1^+/+ and Opa1^−/− IL-2 T_E cells. Each lane represents separate mice with a technical replicate. (D) EM analysis of mitochondrial cristae from T_E and T_M cells isolated after LmOVA infection and (E) in vitro cultured IL-2 T_E and T_M cells. Representative of 2 experiments, scale bar = 0.25 µm. Relative proton leak (ΔOCR after oligomycin and subsequent injection of rotenone plus antimycin A) of (F) Opa1^+/+ and Opa1^−/− IL-2 T_E, (G) infection elicited T_E and T_M, and (H) IL-2 T_E and IL-15 T_M cells. (F–H) Combined from 2–4 experiments shown as mean ± SEM (p**<0.005, ***p<0.0001). (I) EM analysis of IL-15 T_M cell-mitochondrial cristae before and after αCD3/CD28 bead stimulation over hours. Scale bar = 0.2 µm, represents one experiment. (J) Immunoblot analysis of Calnexin and ETC complexes (CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTC01, CV-ATP5A). Equivalent numbers of IL-2 T_E and IL-15 T_M cells lysed in native lysis buffer followed by digitonin solubilization of intracellular membranes with pellet (P) and solubilized supernatant (S) fractions resolved on a denaturing gel, representative of 2 experiments. (K) IL-15 T_M cell, (L) BM-DCs and BM-Macs % ECAR measured at baseline and after media, αCD3/CD28 bead, LPS, or LPS+IFN-γ injection as indicated. Data baselined prior to or right after injection with stimuli. (M) BM-Macs stained for intracellular Nos2 protein by flow cytometry with MFI values (left) and representative histogram (right). (K–M) Shown as mean ± SEM and represent 2–3 experiments (***p<0.0001). See also Figure S6.