Requests for resources and reagents and further information on protocols and experimental designs should be directed to and will be fulfilled by the Lead Contact, Christoph Hess ( chess@uhbs.ch ).

C57BL/6J mice were obtained from Jackson Laboratories. T cell specific Rictor-deficient mice were generated by crossing CD4 promoter controlled Cre recombinase transgenic mice with Rictor-deficient mice. T cell specific Rictor flox/flox : Cd4-Cre + mice were further crossed with OT-I TCR transgenic mice. Rictor −/− strains were backcrossed to the C57BL/6J background. Male and female mice between 16-24 weeks of age and same sex littermates were used for experiments. Mice were maintained at the specific pathogen free animal facilities at the University of Lausanne and at the University of Basel. All animal experiments were performed in accordance with local and Swiss federal guidelines.

Method Details

Human CD8+ T cell isolation and sorting Peripheral blood mononuclear cells (PBMC) were isolated from male and female healthy donors by standard density-gradient centrifugation protocols (Lymphoprep Fresenius Kabi, Norway). CD8+ T cells were enriched by positive selection using magnetic CD8+ beads (Miltenyi Biotec, Germany). Cells were rested overnight prior to cell sorting in RPMI-1640 medium (GIBCO, USA) containing 10% fetal bovine serum (FBS, GIBCO, USA), 50U/ml penicillin and 50 μg/ml streptomycin (GIBCO, USA) (R10FBS). For sorting of naive and EM CD8+ T cells, CD8+ were stained with anti-CD62L (ImmunoTools, Germany) and anti-CD45RA (Beckman Coulter, USA) antibodies. NV and EM CD8+ T cells were identified as CD62L+ CD45RA+ and CD62L– CD45RA– populations, respectively. Cell sorting was performed with a BD influx cell sorter (BD Bioscience, USA). Cells were rested in R10FBS for 2-4 hours at 37°C prior to further experiments.

Mouse T cell isolation and culture Spleen and lymph nodes were removed from OT-I wt and Rictor–/– mice. Single cell suspensions from both tissues were activated with OVA peptide for 3 d and then cultured for an additional 3 d in IL-15 (10 ng/ml). Mouse OT-I cells were cultured in RPMI 1640 containing 10% FCS, 100 U/mL penicillin, 100 μg streptomycin, 0.29 mg/mL L-glutamine, 50 μM β-Mercaptoethanol. In vivo derived, OT-I memory T cells were isolated from L. monocytogenes infected mice (> 40 days post-infection) that were transferred with naive OT-I cells 24 hours before infection. T cells were isolated from splenocytes, and memory OT-I cells sorted using a BD influx cell sorter (BD Bioscience, USA).

T cell activation Human CD8+ T cell activation was performed using in house generated anti-CD3/anti-CD28 coated microbeads unless indicated otherwise. Polybead microspheres (4.5 μm, Polyscience Eppenheim) were incubated with 10 μg anti-human CD28 IgG1 antibody (CD28.2 Biolegend, USA) and 1.5 μg anti-human CD3 IgG2a antibody (HIT3a, Biolegend, USA). Antibody coupling was tested using 2nd antibodies against IgG1 or IgG2a (SouthernBiotech, USA), respectively. Quality control was performed by measuring the amount of antibody molecules per bead and using Quantum MESF microspheres (Bangslab, USA) for reference. All measurements were performed using a BD AccuriC6 flow cytometer (Becton Dickinson, USA) and data analyzed with FlowJo 10.0.8 software (Tree Star, USA). All bead based T cell activations were performed using a 2:1 bead to cell ratio. For 12 hour activation of human T cells used in metabolic flux assays, cells were activated with plate bound anti-CD3 (5 μg/ml, HIT3A) and soluble anti-CD28 (10 μg/ml) antibodies. For direct measurement of activation induced glycolytic switch by metabolic flux analysis, activation beads (5:1 bead to cell ratio) were directly added through the injection port. For in vitro reactivation of mouse T cells, memory OT-I cells were re-stimulated with 10 μM peptide ligands (OVA, R7, G4) for 1 hour (cell fractionation for immunoblots), or 4 hours (intracellular cytokine staining).

Adoptive transfer and bacterial infection 1x105 in vitro generated memory OT-I cells from wt or rictor KO mice were adoptively transferred intravenously (i.v.) into C57BL/6 mice. Recipient mice were then infected with 5x105 CFU virulent LmOVA-N4 at 24 hours post-transfer. Mice were bled at 4 hours post-infection to measure serum cytokines and sacrificed at 24 hours post-infection. Spleens and livers were removed and both organs were homogenized in 0.5% Tergitol/PBS with a Tissuelyser (QIAGEN). Organ suspensions were plated on brain-heart-infusion (BHI) and bacterial colonies counted at 24 hours after plating.

Metabolic assays A Seahorse XF-96e extracellular flux analyzer (Seahorse Bioscience, Agilent) was used to determine the metabolic profile of cells. NV and EM CD8+ T cells were plated (3x105 cells/well) onto Celltak (Corning, USA) coated cell plates. Mitochondrial perturbation experiments were carried out by sequential addition of oligomycin (1 μM, Sigma), FCCP (2 μM, Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, Sigma), and rotenone (1 μM, Sigma). Oxygen consumption rates (OCR, pmol/min) and extracellular acidification rates (ECAR, mpH/min) were monitored in real time after injection of each compound. For monitoring oxygen consumption in permeabilized cells, Jurkat and CD8+ T cells were resuspended in MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 2 mM HEPES, and 1 mM EGTA), then treated with the XF plasma membrane permeabilizer (Seahorse, Agilent), which was followed by treatment with pyruvate (5 mM)/malate (2.5 mM)/ADP (4 mM), or pyruvate/malate/oligomycin (2 μM) for monitoring State III ADP respiration and non-ATP synthase dependent respiration, respectively. ATP quantitation (Abcam) was performed on 2.5x104 non-activated and activated T cells according to the manufacturer’s instructions.

IFN-γ measurement EM CD8+ T cells were plated on flat-bottom 96 well plates (3x105 cells/well) using R10FBS. In select experiments, cells were pre-incubated for 30 min. with inhibitors and then activated using anti-CD3/anti-CD28 loaded beads. Cell supernatants were harvested 12 hours after activation and IFN-γ was measured using either an IFN-γ ELISA kit (eBioscience, USA) or a human Th1 cytokine bead-based immunoassay (Legendplex, Biolegend) according to manufacterer’s instructions. For cytokine measurements in mouse sera, a bead-based immunoassay was also used.

Metabolomics NV and EM CD8+ T cells were cultured for 2 hours in a 24 well plate (3x106 cells/well) under non-activating and activating conditions. For Akt inhibition, cells were treated with Akti1/2 (10 μM, Sigma-Aldrich). Cell pellets were washed twice with cold PBS, snap frozen in EtOH containing dry ice and stored at −80°C. Metabolomic assays and analysis were performed by Metabolon Inc. (Durham, USA).

Flow cytometry For cell proliferation assays, EM CD8+ T cells were loaded prior to activation with the cell-proliferation dye carboxyfluorescein succinimidyl ester (1 μM CFSE, Molecular probes, USA) and seeded on 48-well plates (5x105 cells/well). Plated cells were pretreated with inhibitors as indicated, and cultured with activation beads and IL-2 (1000 U/ml) (ImmunoTools, Germany) for 3 days. For surface staining of CD8+ T cells, human T cells were stained with antibodies targeting CD8, CD3, CD45RA, CD62L, CD69, and mouse OT-I cells were stained with antibodies targeting CD69, CD44, CD127, CXCR3, Ly6C, CD62L, KLRG1, CD27, CD25, CD69, CX3CR1, and CCR7. For intracellular IFN-γ measurements, cells were treated with Brefeldin A after 2 hours of activation and stained using a standard intracellular cytokine staining protocol (BD Bioscience). For measurement of mitochondrial mass and membrane potential, cells were stained with mitotracker green (MTG, 25 nM) or mitotracker red (MTR, 25 nM), respectively. Data was acquired using a BD AccuriC6 flow cytometer and analyzed with Flowjo 10.0.8 (Tree Star, USA).

Total cell lysate preparation and cell fractionation To prepare total cell lysates, cells were activated for 1 hour (beads or peptide). Cell pellets were washed with cold PBS and lysed using RIPA buffer (Thermo Scientific, Rockford IL, USA) containing protease and phosphatase inhibitors (Roche). To prepare mitochondrial fractions that are enriched in mitochondria–ER junctions, cells were washed and resuspended in homogenization buffer (225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris–HCl pH 7.4), and homogenized with 20 strokes (CD8+ T cells) or 50 strokes (Jurkat T cells) using a teflon homogenizer. Cell homogenates were centrifuged twice at 600xg for 5 min. (4°C) and pellets were discarded after each spin. Supernatants were then centrifuged at 10’300xg, 10 min. (4°C). Supernatants (containing ER, Golgi and cytoplasm) were collected, and pellets (containing mitochondria–ER junction-enriched mitochondria) were resuspended in RIPA buffer (Thermo Scientific, Rockford IL, USA). A digitonin-based protocol using glass homogenizers was used to prepare pure mitochondrial fractions.

Immunoblots Protein concentrations were determined by BCA protein assay kit (Thermo Scientific). Total cell lysates, cytoplasmic, mitochondria–ER junction-enriched mitochondrial fractions or pure mitochondrial fractions were separated using 4%–15% Mini Protean TGX Gel (Bio-Rad, Hercules CA, USA), and transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer (Bio-Rad, Hercules CA, USA). Membranes were probed with antibodies against ACC, rictor, mTOR, HK-I, HK-II, Sin1, Cox iv, Akt, pAkt Thr308, pAkt Ser473, p-(S/T) Akt substrate motif, pGsk-3β Ser9 (all from cell signaling), GRP75, VDAC1, Gsk-3β (from Abcam), actin (Sigma-Aldrich), or raptor (Bethyl). Blots were stained with appropriate secondary antibodies and the odyssey imaging system (LICOR, Lincoln NE, USA) was used for visualization.

Electron microscopy Transmission electron microscopy was performed at the Biozentrum (University of Basel). Cells were sequentially fixed in 3% paraformaldehyde, 0.5% glutaraldehyde and 1% osmium tetraoxide, embedded and then cut into 60 nm sections. Micrographs (27’000x magnification) were obtained with a Morgagni 268 (FEI, Hillsboro OR, USA) transmission electron microscope operated at 80 kV. ImageJ software (NIH, Bethesda, USA) was used for measuring mitochondrial length (major axis) and width (minor axis). To quantify the morphology of mitochondria, the average aspect ratio was calculated (major axis to minor axis), an aspect ratio of 1 indicates a circular mitochondrial section. To quantify mitochondria–ER contacts for each cell, total mitochondria and mitochondria in contact with ER were counted and the frequency of mitochondria–ER contacts calculated. Mitochondria–ER contacts were defined as mitochondria juxtaposed to ER at a distance of < 100 nM.

Proximity Ligation Assay Anti-VDAC1 and anti-IP3R1 antibodies were used to visualize mitochondria–ER junctions. T cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% goat serum. PLA was performed according to the manufacturer’s instructions (Duolink, Sigma). Fluorescent spots were detected by using an inverted spinning disk confocal microscope, Visiscope CSU-W1 (Visitron). Quantification of detected spots per nucleus was performed by using the ImageJ software.

RNA mediated interference Jurkat T cells were transfected with pools of Grp75 siRNA or control scrambled siRNA (QIAGEN) for 72 hours using the AMAXA T cell nucleofection kit (Lonza). Knockdown efficiency was verified by immunoblot analysis.

Confocal imaging Experiments with fixed cells were performed on a Nikon A1 confocal microscope (Microscopy core facility, DBM) with a 40x oil-immersion objective. Cells were allowed to attach by gravity on culture slides (BD Bioscience) coated with poly-d-lysine for 20 min. Cells were pre-stained with mitotracker deep red and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were stained with antibodies against rictor (Atlas antibodies), KDEL motif (Abcam) or Gsk-3β (Abcam), labeled with the appropriate secondary antibodies, and counterstained with DAPI. All images were processed and analyzed using ImageJ (NIH). Experiments with live cells were performed using the 3I spinning disk confocal microscope (Intelligent Imaging Innovations, Denver CO, USA) with a 100x oil-immersion objective at 5% CO2 and 37°C (IMCF Facility, Biozentum, University of Basel). Cells were loaded with 50 nM MTG (Invitrogen) and 25 nM tetramethylrhodamine-ethyl-ester-perchlorate (TMRE, Invitrogen). Cells were incubated for 30 min. with both dyes, washed and resuspended in fresh media. Since CD8+ T cells are non-adherent, dye-loaded T cells were allowed to settle onto culture slides (Ibidi, Munich, Germany) coated with poly-d-lysine for 20-30 min. before imaging. Stacked images were then acquired at 0.5 μm intervals throughout the body of each cell. Quantification of mitochondrial structure was carried out on cells labeled with MTG. Extraction of mitochondrial structural parameters was performed using a multi-step image-processing algorithm. Briefly, MTG intensities in raw images were enhanced by linear contrast stretch, spatially process using a 7x7 top hat filter to isolate mitochondrial objects and converted into a binary image by a threshold operation. Mitochondrial circularity (4π area/perimeter2), length, and form factor (1/circularity) were then calculated using ImageJ (NIH, Bethesda MD). Mitochondrial membrane potential (Δψ m ) was calculated as the ratio of TMRE to MTG fluorescence intensities. MTG was included to normalize for mitochondrial mass differences between cell populations.