and C57/BL6 mice were purchased from the Jackson Laboratory. Cd4
-Cre mice were purchased from Taconic. Scapflox/flox
mice were crossed with Cd4
-Cre mice to generate Cd4
mice were used as littermate control for all experiments using Cd4
mice. All mice were maintained in pathogen free facilities of the University of California, Los Angeles. All experiments on mice and tissues collected from mice were performed in strict accordance with University of California, Los Angeles policy on the humane and ethical treatment of animals.
Cell culture and reagents
Cells were cultured in IMDM supplemented with 10% heat inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 uM 2-ME. Total T cells or CD8+ T cells were purified using negative enrichment kit (STEMCELL Technologies). T cells were cultured in 96-well plates coated with 5 μg/ml of anti-CD3 (2C11;Bio X Cell) alone or in combination with 2 μg/ml of soluble anti-CD28 (37.51;Bio X Cell) or 100 IU/ml of human IL-2. Alternatively, cells were cultured with 50 ng/ml of PMA (EMD Chemicals) alone or in combination with 500 ng/ml of ionomycin (Sigma-Aldrich). For proximal TCR signaling analysis, splenocytes were cultured with 1 μg/ml of soluble anti-CD3. In some experiments, cells were cultured with following inhibitors for 30 min prior to activation: Gö6983 (Sigma-Aldrich), Ly294002 (EMD Chemicals), Rapamycin (EMD Chemicals), 25-hydroxycholesterol (Sigma-Aldrich).
Truncated forms of SREBP1a or SREBP2 (Supplementary Fig. 1c
) were cloned into the retrovirus vector MIGR1 (a gift from Dr. Yukiko Tone and Dr. Masahide Tone, Cedars-Sinai, Los Angeles, USA). Virions were produced using the 293T cell line by transfection using Lipofectamine 2000 (Invitrogen). Purified naiveT cells were stimulated in 6-well plates pre-coated with 5 μg/ml of anti-CD3 supplemented with 2 μg/ml of anti-CD28 at 2 × 106
cells/well. At 24 hours, cells were transduced by centrifugation in viral medium plus 8 μg/ml of polybrene and 100 IU/ml of IL-2 at 1200 rpm for 90 min at 37 °C. Medium was then replaced with complete IMDM supplemented with 100 IU/ml of IL-2 and cells were incubated for additional 24 hours. Control cells were subjected to the same stimulation and centrifugation as transduced cells but without virus. After a total of 48 hours of stimulation, cells were harvested and transduction efficiencies were evaluated by GFP expression level on flow cytometry.
Purified T cells were transfected with non-target siRNA, SREBP1 siRNA, or SREBP2 siRNA (Dharmacon) using Nucleofector according to manufacture’s protocol (Lonza). Transfected cells were rested for 18 hours at 37 °C and then stimulated with PMA and ionomycin for 5 hours.
Quantitative real-time PCR analysis
RNA was isolated using Trizol (Invitrogen). cDNA was synthesized using iScript cDNA Synthesis Kit (BIO-RAD). qRT-PCR was performed using SYBR Green I Master (Roche) on LightCycler 480 (Roche). Primer sequences are available upon request.
Flow cytometry analysis
7-ADD, anti-mouse CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61.5), CD44 (IM7), Thy1.2 (53-2.1), IL-7R (CD127), KLRG1 (2F1), INFγ (XMG1.2), TNFα (MP6-XT22), and IL-2 (JES6-5H4) were purchased from eBioscience, BioLegend, or BD Biosciences. Active caspase3 and annexin V staining kits were purchased from BD Bioscience. CFDA SE, ER-Tracker Blue-White DPX, and DAPI were purchased from molecular probe. Propidium Iodide was purchased from Calbiochem. Cells were analyzed on LSRII or FACSVersa with FlowJo software (Treestar).
Whole cell extracts were produced from equivalent number of cells with RIPA buffer (50mM Tris-HCl [pH 8.0], 150mM NaCl, 1% NP40, 0.5% Deoxycholate, 0.1% SDS) supplemented with Calyculin A (Cell Signaling) and protease inhibitor cocktail (Sigma-Aldrich). Samples were separated on a 4-12% Bis-Tris gel and transferred to nitrocellulose. Antibodies for phospho-Akt (S473), Akt1 (5C10), Actin (I-19) were purchased from Santa Cruz Biotechnology. Antibody for HIF1α was from Cayman Chemical. Antibodies for S6 ribosomal protein (54D2), phospho-S6 (S235/236)(D57.2.2E), Rb (D20), phospho-Rb (Ser807/811), p27Kip1, Cyclin D2 (D52F9), Cyclin D3 (DCS22), MYC, phospho-Zap70 (Tyr319)/Syk (Tyr352)(65E4), phospho-Lck (Tyr505), LAT, phospho-LAT (Tyr191), p44/42 MAPK (137F5), phospho-p44/42 MAPK (Thr202/Tyr204)(D13.14.4E), AMPK (F6), phospho-AMPK (Thr172)(40H9), phospho-ACC (Ser79) were from Cell Signaling.
Chromatin immunoprecipitation analysis
The method is described elsewhere34
. Briefly, 2×107
cells were fixed and sonicated. Precleared lysates were incubated overnight at 4 °C with polyclonal anti-SREBP1 and anti-SREBP2, or control rabbit IgG (Cell Signaling). Immunocomplex was collected and real-time PCR was performed as described above. Primer sequences are available upon request.
Gene expression analysis
RNA isolated from littermate control and Scap
T cells and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Array. Genes were filtered for differential expression across the samples by requiring an ANOVA p-value less than 0.001. For heatmap display, genes were mean centered, normalized and clustered using a Pearson correlation coefficient metric and pairwise complete-linkage. Pathway enrichment analysis was done with DAVID 20, 21
. Transcription factor analysis was generated through the use of IPA (Ingenuity Systems, www.ingenuity.com
Cellular ATP levels were measured from the same number of live cells using ATP determination kit according to the manufacture’s protocol (Molecular Probes). Background was measured using buffer without cells and subtracted from the values of each cell sample. For mitochondrial mass, cells were incubated with 50 nM MitoTracker green (Molecular Probes) at 37 °C for 30 min. The oxygen consumption rate and extracellular acidification rate were measured with a XF24 analyzer according to the manufacture’s recommendations 35
. Mixing, waiting and measure times were 2, 2, 4 minutes, respectively. Test compounds were obtained from Sigma and injected during the assay at the following final concentration: 0.2 μM oligomycin, 1 μM FCCP, 0.75 μM Rotenone/Myxothiazol. Glucose, glutamine, and lactate concentrations in culture media were measured using bioprofile analyzer (Nova Biomedical).
Cholesterol:MβCD complex containing 40 mg of cholesterol/g was purchased from Sigma (C4951). Treatment concentration was based on cholesterol weight. Cells were activated with or without cholesterol:MβCD in 10% FBS for indicated time.
Purified T cells were collected at ex vivo
or after 24 hours of activation with plate-bound anti-CD3 in combination with soluble CD28 and washed twice with PBS. Fatty acids extractions and analyses were performed as previously described 36
with the modifications described below. Triheptadecanoin (Nu-chek Prep, T-155) was used as the internal standard for fatty acids. Ergosterol (Sigma 17130-U) was used as the internal standard for cholesterol. Internal standards were added to trizol. Cholesterol was extracted from organic phase with petroleum ether after saponification but before acidification and fatty acid extraction. Extracted cholesterol was trimethylsilylated with BSTFA and TMCS (Sigma 33155-U). Extracted fatty acids were derivatized into their methyl ester form with methanolic boron trifluoride. Derivatized cholesterol and ergosterol were monitored at m/z 456-467 and 468 respectively. Derivatized myristate, palmatate, heptadecanoate, and sterate were monitored at m/z 241-250, 269-278, 284, and 297-306 respectively. Data was collected on an Agilent 5975C MSD connected to an Agilent 7890 Gas Chromatograph with the Phenomenex ZB-MR-1 column. Settings and oven programs are available upon request. Area under the curve quantitation for selected ions was performed on Chemstation software.
LCMV infection, MHC I tetramer and intracellular cytokine staining
Mice were infected intraperitoneally (i.p.) with 2 × 104
plaque forming units (PFU) of LCMV-Armstrong. Virus stocks were prepared and viral titers were quantified as described previously 37
. Splenocytes were stained directly ex vivo with LCMV-Db
specific tetramers and for surface expression of CD8. MHC tetramers were obtained from the NIH Tetramer Core Facility. To analyze cytokine expression, splenocytes were stimulated for 5 hours with 2 μg/ml of the MHC class I restricted LCMV-GP33-41
peptide in the presence of 50 U/ml recombinant murine IL-2 (R&D Systems) and 1mg/ml brefeldin A (Sigma-Aldrich). Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend). Flow cytometric analysis was performed using an LSR II or the FACSVerse (Becton Dickinson) and analyzed using FlowJo software (Treestar).
Adoptive cell transfer
CD8+ T cells or total T cells were purified from Scapfl/fl and control littermate mice or Thy1.1 mice spleens as above. Purified cells were labeled with CFSE and 1-5 × 106 cells/mouse were injected via retro-orbital into RAG-deficient host or irradiated host 1 day earlier with 600 rad. After 6 days, host spleen and LN cells were analyzed by flow cytometry. For gene expression analysis, Thy1.1+ T cells were sorted and real-time PCR was performed as described above.
OVA peptide-IFA immunization
Forty-eight hours after the OT-I cell transfer, mice were either left unimmunized or immunized with total of 300 μg of OVA peptide mixed in IFA into 3 sites in upper back and 3 sites in lower back (n=3 per group). Spleen and lymph nodes were harvested 24 hours later and CD3+CD8+ cells were analyzed on flow cytometry.
BrdU flow kit was purchased from BD Bioscience. BrdU (2 mg/mouse) was injected to Cd4-Cre/Scapflox/flox and littermate control mice intraperitoneally. Peripheral blood was collected twice a week and mononuclear cells were stained and analyzed on flow cytometry according to manufacture’s protocol.
Statistical significance was determined with the two-tailed unpaired Student’s t-test unless otherwise indicated.