METHODS DETAILS

CD4⁺ T cells and total T cells were isolated from single cell suspensions of spleens and lymph nodes by negative separation using a mouse CD4⁺ T cell or a total T cell Enrichment kit (both STEMCELL Technologies). T cells were stimulated in RPMI 1640 medium (supplemented with 10% FBS, 2 mM L-glutamine, 50 mM 2-mercaptoethanol and 100 U/ml penicillin plus streptomycin; all Cellgro) with 1 μg/ml plate-bound anti-CD3 (clone 2C11) plus 1 μg/ml anti-CD28 Abs (clone 37.51, both BioXCess) in the presence or absence of 1 μM FK506 (Sigma Aldrich), 10 mM 2-DG (Sigma Aldrich), 5 ng/ml IL-7 (Peprotech), 50 U/ml IL-2 (Peprotech) or left unstimulated as indicated. PBMCs from individuals homozygous (patient) and heterozygous (mother) for a STIM1 p.L374P mutation were isolated from blood samples by density centrifugation using Ficoll-Paque plus (GE Amersham). Human CD4⁺ T cells were isolated using CD4 MicroBeads (Miltenyi Biotec). PBMCs or purified T cells were stimulated with 1 μg/ml plate-bound anti-CD3 (clone OKT3) and 1 μg/ml anti-CD28 (clone CD28.2, both eBioscience) monoclonal antibodies in the presence or absence of 1 μM FK506 (Sigma Aldrich) or 500 nM BTP2 (Sigma Aldrich) or left unstimulated as indicated.

The murine Slc2a3 promoter (-475/+358) was amplified from genomic DNA using the primers 5′-AAAAGCTTGAAATGTGCCTGCCTCCTGC-3′ and 5′-TTGTCCCCATGGTCCCAACC-3′ and cloned as a HindIII/NcoI fragment matching Slc2a3-ATG with luciferase ATG into the pGL3-Basic backbone (Invitrogen). The -16 kb distal enhancer region of the Slc2a3 gene was amplified by PCR using the primers 5′-AAAAGCTTGAAATGTGCCTGCCTCCTGC-3′ and 5′-TTGTCCCCATGGTCCCAACC-3′ and cloned as a 916 bp NheI/XhoI fragment upstream of the promoter. HEK 293T cell reporter gene assays were performed as described (Vaeth et al., 2014).

Vectors encoding constitutively active NFATc1 (MIGR-caNFATc1-IRES-GFP) (Gomez-Rodriguez et al., 2009) and GLUT1 (Slc2a1, pMIGR-Slc2a1-IRES-VEX) were kindly provided by P. Schwartzberg (NIH Human Genome Research Institute, Bethesda, MD) and J. Wherry (University of Pennsylvania, Philadelphia, PA), respectively. 20 bp oligonucleotides targeting exon 1 of the Slc2a3 gene (5′-GGGCGTTCTTCAATGCCACG-3′, 5′-CATTGTGTGGAGTTTTGCCG-3′, 5′-TGTGTTGTAGCCAAACTGCA-3′) were cloned into a retroviral vector for guide RNA delivery, which was a kind gift from M. Aichinger and J. Zuber (Vienna Biocenter, Austria). Expression vectors for IRF4 (pMIG-IRF4, #58987) c-Myc (pMIG-Myc, #58987) were obtained from Addgene. anti-CD4 (clone GK1.5), anti-CD8 (53-6.7), anti-TCR Vα2 (B20.1), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-Ki-67 (SolA15) antibodies were from eBioscience. Mitochondrial complexes were detected with the Total Oxphos Rodent WB antibody cocktail (MitoSciences) and an HRP-conjugated anti-mouse secondary antibody (Sigma Aldrich). GLUT1 protein expression was detected with a polyclonal anti-GLUT1 antibody (Abcam, ab115730) and an Alexa-647 conjugated goat-anti-rabbit-IgG secondary antibody (Molecular Probes). GLUT3 expression was analyzed using a polyclonal, FITC-conjugated anti-mouse-GLUT3 antibody (Abcam, ab136180). For analysis of phosphorylated AKT, mTOR and ribosomal S6 protein, total T cells were stimulated for 24 h with anti-CD3/CD28 with or without 1 μM FK506 and immediately fixed with IC fixation buffer (eBioscience). Protein phosphorylation was detected in permeabilized CD4⁺ and CD8⁺ T cells using anti-phospho-AKT Thr308 (D25E6, Cell Signaling Technology), anti-phospho-AKT Ser473 (SDRNR), anti-phospho-mTOR Ser2448 (MRRBY) and anti-phospho-S6 Ser235/236 (cupk43k, all eBioscience). Unconjugated rabbit anti-phospho-AKT Thr308 primary antibody was detected using an AlexaFlour-647-conjugated goat-anti-rabbit IgG secondary antibody (Molecular probes).

CD4⁺ T cells were isolated using a mouse CD4 T Cell Enrichment kit (STEMCELL Technologies) and stimulated with 1 μg/ml plate-bound anti-CD3 (clone 2C11) plus 1 μg/ml anti-CD28 Abs (clone 37.51, both BioXCess) in the presence of 50 U/ml IL-2 (Peprotech) and 2.5 ng/ml IL-7 (Peprotech). T cells were transduced 24 hours after stimulation by spin-infection (2.500 rpm, 30°C, 90 min) in the presence of concentrated retroviral supernatant and 10 μg/ml polybrene (SantaCruz). Retroviral supernatant was produced in the Platinum-E retroviral packaging cell line. Platinum-E cells were transfected by lipofection (GeneJet, Fisher) with retroviral expression plasmids and the amphotrophic packaging vector pCL-10A1. Two days after transfection, the supernatant was collected and concentrated using Amicon Ultra-15 centrifugal filters (Merck Millipore). 4 hours after spin-infection, viral supernatant was removed from the T cells and replaced by fresh media. 48 h after transduction, T cells were transferred into new plates adding fresh media containing 50 U/ml IL-2 and 2.5 ng/ml IL-7 and rested for 2 days in IL-2 and IL-7 containing medium. Transduced cells (GFP⁺) were FACS-sorted using a sterile Sony SY3200 (HAPS1) cell sorter and 4x10⁵ GFP⁺CD45.1⁺ SMARTA CD4⁺ T cells were retro-orbitally injected into recipient CD45.2⁺ host mice. 3 days after adoptive transfer, host mice were infected i.p. with 2x10⁵ PFU LCMV (Armstrong strain) and expansion of CD45.1⁺ Va2 TCR⁺ donor T cells in the spleen was analyzed 8–10 days later by flow cytometry as described (Vaeth et al., 2016).

Cells were washed in ice-cold PBS containing 1% FBS before blocking with anti-FcγRII/FcγRIII antibodies (2.4G2, eBioscience). Staining of surface molecules with fluorescently labeled antibodies was performed at room temperature for 20 min in the dark. GLUT1 or GLUT3 expression was detected in cells fixed and permeabilized with the IC Staining Buffer Kit (BioLegend) using a polyclonal rabbit anti-GLUT1 antibody (Abcam, ab115730) together with an Alexa-647 conjugated anti-rabbit IgG secondary antibody (Molecular Probes) or a polyclonal, FITC-conjugated anti-GLUT3 antibody (Abcam, ab136180). Samples were acquired on a LSRII flow cytometer using FACSDiva software (BD Biosciences) and further analyzed with FlowJo software (Tree Star).

CD4⁺ and total T cells were loaded with 2.5 μM CFSE (Molecular Probes) according to the manufacturer’s instructions and stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of 1 μM FK506, 50 U/ml IL-2, 5 ng/ml IL-7, a combination of IL-2 and IL-7 or left unstimulated as indicated. 72 to 96 h after stimulation, CFSE dilution was assessed by flow cytometry.

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using an XF^e24 Extracellular Flux Analyzer (Seahorse Bioscience). Before experiments, cells were resuspended in XF media (Seahorse Biosciences) supplemented with 10 mM glucose (Sigma Aldrich), 1 mM GlutaMAX (Gibco) and 1 mM sodium pyruvate (Corning) and analyzed under basal conditions and following treatment with the following agents: the ATP synthase inhibitor oligomycin (1 μM); the protonophore Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.75 μM) to uncouple mitochondria; the mitochondrial complex I inhibitor rotenone (100 nM) and the mitochondrial complex III inhibitor antimycin A ( 1 μM). The basal oxygen consumption rate (OCR) was calculated by subtracting the OCR after rotenone and antimycin A treatment from the OCR before oligomycin treatment. The maximal OCR was calculated by subtracting the OCR after rotenone and antimycin A treatment from the OCR measured after addition of FCCP.

LC/MS analyses were conducted on a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe, which was coupled to a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific, San Jose, CA). External mass calibration was performed using the standard calibration mixture every 7 days. For metabolite profiling experiments, CD4⁺ T cells were stimulated with plate bound anti-CD3 and anti-CD28 for 36 hours. Cells were collected and cell pellets were washed with ice cold PBS. Polar metabolites were extracted using 1 ml of ice-cold 80% methanol with 10 ng/ml valine-d₈ as an internal standard. After a 10 min vortex and centrifugation for 10 min at 4°C at 10,000 g, samples were dried in a table-top vacuum centrifuge. Dried samples were stored at −80°C and then resuspended in 100 μl water; 1 μl of each sample was injected onto a ZIC-pHILIC 2.1 x 150 mm (5 μm particle size) column (EMD Millipore). Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide; buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.150 ml/min as follows: 0–20 min.: linear gradient from 80% to 20% B; 20–20.5 min.: linear gradient from 20% to 80% B; 20.5–28 min.: hold at 80% B. The mass spectrometer was operated in full-scan, polarity switching mode with the spray voltage set to 3.0 kV, the heated capillary held at 275°C, and the HES I probe held at 350°C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 1 unit. The MS data acquisition was performed in a range of 70–1000 m/z, with the resolution set at 70,000, the AGC target at 10⁶, and the maximum injection time at 80 msec. Relative quantitation of polar metabolites was performed with XCalibur QuanBrowser 2.2 (Thermo Fisher Scientific) using a 5 ppm mass tolerance and referencing an in-house library of chemical standards. Relative abundance of metabolites was calculated by normalizing profiling results to cell number.

Total DNA from cells was isolated using the FlexiGene DNA kit (Qiagen). Oligonucleotide probes were designed against 3 different regions of mitochondrial DNA (mtDNA), and 2 regions of genomic DNA. Specific primers used are listed in Table S1. Quantitative realtime PCR was performed using the Maxima SYBR Green qPCR Master Mix (Thermo). mtDNA copy number was calculated relative to genomic DNA as previously described (Maus et al., 2017).

Glucose utilization was measured 48 hours after stimulation by quantifying the glucose content of RPMI 1640 media (supplemented with 10% FBS, 2 mM L-glutamine, 50 mM 2-mercaptoethanol and 100 U/ml penicillin and streptomycin; all Cellgro) with the colorimetric Glucose (HK) assay (Sigma Aldrich) in a 96 well microplate reader (Molecular Devices). Utilized glucose was calculated by subtracting the glucose content of the cell culture media after 48 h from the glucose content of fresh media and normalized to cell number. Glucose uptake was analyzed directly using the fluorescent glucose analogue 2-NBDG (ThermoFisher Scientific). Stimulated and unstimulated cells were incubated in glucose-free RPMI medium containing 100 μM 2-NBDG for 90 minutes at 37°C and the amount of 2-NBDG taken up by cells was assessed by flow cytometry. Alternatively, glucose uptake was measured using tritiated (³H) 2-DG (PerkinElmer). CD4⁺ T cells were stimulated with anti-CD3/CD28 (both at 1 ug/ml and plate-bound) for 48 h in the presence or absence of 1 μM FK506. 4x10⁶ T cells were incubated at 37 °C in 1 ml glucose-free medium (with 10% FBS) containing 1 μCi ³H-2-DG for 8 h. After washing twice with ice-cold PBS, cells were resuspended in 1 ml PBS, counted and homogenized using 4 ml Ultima Gold scintillation cocktail (PerkinElmer). Intracellular ³H-2-DG was measured with an automated LS 6500 scintillation counter (Beckman Coulter). ³H counts per minute (cpm) were normalized to cell number.

T cells were stimulated for 36 h with anti-CD3 and anti-CD28 in the presence or absence of 1 μM FK506 and harvested on slides using a Shandon cytospin centrifuge (400 rpm for 4 min, RT). After drying o/n, cells were fixed with 4% PFA in PBS for 20 min, washed 3 times, permeabilized with 0.2% Triton-X for 5 min and washed with PBS. Nonspecific binding was blocked with antibody diluent (DAKO) for 60 min as described (Vaeth et al., 2011). NFATc1 intracellular localization was detected with a monoclonal anti-NFATc1 primary antibody (1:100 dilution; clone 7A6, ab2796, Abcam) incubated with cells o/n at 4 °C followed by incubation with an AlexaFluor 555-conjugated donkey-anti-mouse IgG secondary antibody (1:800 dilution; Molecular Probes) for 1h at RT. After washing, slides were mounted in Fluoromount G (Southern Biotechnology) containing DAPI and analyzed on a Leica TCS SP5 II confocal microscope. Data was processed using the LCS software package (Leica) and ImageJ (NIH). For statistics, >100 cells from 3 mice were quantified for cytosolic/nuclear localization of NFATc1.

Isolated human CD4⁺ T cells were bound to poly-L-Lysine (Sigma Aldrich) coated translucent 96-well plates (BD Falcon). Cells were loaded with 1 μM Fura-2-AM (Molecular Probes), and washed twice with 0 mM Ca²⁺ Ringer solution as described (Feske et al., 2006; Vaeth et al., 2017). Ca²⁺ measurements were performed using a FlexStation 3 multi-mode microplate reader (Molecular Devices). Cells were resuspended in 0 mM Ca²⁺ Ringer solution at the beginning of measurements. After 120 seconds, 1 μM thapsigargin (Calbiochem) was added to induce activate CRAC channels by depleting ER Ca²⁺ stores. At 420 seconds, 2 mM Ca²⁺ containing Ringer solution was added to the cells (to obtain a 1 mM final extracellular Ca²⁺ concentration) to measure SOCE. Fura-2 fluorescence was measured at 510 nm after excitation at 340 nm and 380 nm and plotted as the F340/F380 emission ratio.

Total RNA was isolated using Trizol (Invitrogen) or the RNeasy Micro Kit (Quiagen) and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). Quantitative realtime PCR was performed using the Maxima SYBR Green qPCR Master Mix (Thermo) and gene specific primers (Table S1). The relative abundance of transcripts was normalized to the expression of housekeeping genes using the 2^−ΔCT method. Relative expression values were calculated by normalizing gene expression to the average of untreated WT or healthy donor controls.

Total cell lysates were prepared in lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.4), 250 mM NaCl, 10 mM EDTA, 2 mM sodium-o-vanadate, 10 mM sodium pyrophosphate, 10% glycerol, 10 μg/ml aprotinin, 10 μg/ml pepstatin, 5 μg/ml leupeptin and 0.2 mM PMSF. Lysates from equal numbers of cells for each condition were subjected to SDS-PAGE, transferred to nitrocellulose membrane, blocked with low-fat milk and incubated with the Total Oxphos Rodent WB antibody cocktail (MitoSciences) overnight. Proteins were visualized using an HPR-conjugated anti-mouse secondary antibody (Sigma) and chemiluminescent ECL reagent (Thermo Fisher). Densitometric quantification was performed using ImageJ.

NFATc2 ChIP-seq data (Martinez et al., 2015) and genome-wide NFATc1 chromatin binding data (using T cells from a BAC-transgenic mouse strain in which NFATc1 is endogenously biotinylated by the biotin-ligase BirA and precipitated using streptavidin beads) (Klein-Hessling, 2017) were aligned to open chromatin regions defined by DNase I hypersensitivity sites in naïve and CD8⁺ T cell blasts (Bevington et al., 2016) and accessible chromatin regions defined by ATAC-seq in resting and PMA/ionomycin-stimulated CD8⁺ T cells (Mognol et al., 2017). All sequencing data was mapped to the mouse mm9 genome assembly using the Bowtie software package. NFATc1 and NFATc2 binding peaks were identified using the model-based analysis of ChIPseq (MACS) method.