Further information and requests for resources and reagents should be directed to and will be fulfilled according to institutional rules by the Lead Contact, Hildegund Ertl ( ertl@wistar.org ).

The B16Braf V600E cell line was derived from B16.F10 cells by transduction with the lentivector pLU-EF1a-mCherry expressing mouse Braf V600E . The B16 OVA cell line was a gift from Dr. R. Dutton (Wadsworth Center, Albany, NY). HEK 293 cells were used to propagate vaccine vectors. 293T cells were used to produce lentivectors. Cells were grown in Dulbecco’s Modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Human studies were approved by the Institutional Review Board (IRB) of the Wistar Institute (protocol 21111257, protocol 2802240-4b). Informed consent was obtained from each patient before blood or tumor tissue collection. The human blood and serum samples were obtained from clinical research unit, Duke University (Durham, NC, USA) in accordance with the local IRB. Samples were from male and female Caucasians between the ages of 32-39. Lymphocytes from periphery blood were isolated by Ficoll-Paque PLUS (GE Healthcare Biosciences) gradient using standard procedures. The PDX sample and the fresh metastatic tumors from melanoma patients were obtained from melanoma patients at the Hospital of the University of Pennsylvania upon their informed consent under protocol 2802240-4b. To ensure patient confidentiality no information on age, gender or ethnicity were provided to the investigators of this study. Metastases were cut into small pieces, digested with Collagenase/DNAse I and filtered through 70 μm cell strainers to produce single cell suspension. TILs were purified by Percoll-gradient.

For in vivo treatment FF (100 mg/kg/day) in DMSO/PBS was given by oral gavage daily for 3 weeks. Control mice received diluent. For co-transfer experiments, splenocytes containing 10 5 MAA-specific CD8 + T cells from the experimental and control groups were mixed and transferred i.v. into CD90.1 + recipient mice. For FF-treated OT-1 CD8 + T cell transfer, FF was added for 48 hr at 25 μM on day 4 of OT-1 T cell activation. For PD-1 blockade experiments in NSG or unvaccinated C57BL/6 mice, α-PD-1 Ab (clone 29F.1A12) or the isotype control Ab (Iso, Clone: 2A3, Bio X Cell) were given as of day 3 after tumor cell challenge. In vaccinated C57BL/6 mice, α-PD-1 or Iso treatment was started 10 days after vaccination. The Abs were given i.p. every 3 rd day at a dose of 200 μg/mouse.

All procedures were conducted following protocols approved by the Wistar IACUC. Female C57Bl/6, B6.SJL-Ptprc a Pepc b /BoyJ (B6 CD45.1 + ), B6.PL-Thy1 a /CyJ (B6 CD90.1 + ), B6; 129S4-Ppara tm1Gonz /J (B6 PPAR-α KO), C57Bl/6-Tg(TcraTcrb)1100Mjb/J (OT-1) and NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice (6-8 weeks) were purchased from Charles River, National Cancer Institute (NCI) or Jackson Laboratories and housed at the Wistar Institute Animal Facility. Groups of 5-80 C57BL/6 mice were vaccinated intramuscularly (i.m.) with 10 10 virus particles [vp] of AdC68-gDMelapoly, 10 11 vp of AdC68-gDE7 or 10 11 vp AdC68-gD. For tumor challenge experiments B16Braf V600E cells (5x10 4 cells/mouse) or B16 OVA cells (10 5 cells/mouse) were resuspended in PBS and injected subcutaneously (s.c.) into the right flank. For PDX model, NSG mice were injected s.c. with WM4231-2 cells (10 5 cells/mouse). The WM4231-2 cells were established from a surgically removed metastatic lesion from a patient with treatment-naive melanoma. Tumor growth was monitored by measuring the perpendicular diameters of tumors every other day. Depending on size early stage tumors were harvested 10-14 days after challenge (referred to as day 14) while late stage tumors were harvested 4-5 weeks after challenge (referred to as day 30). For adoptive transfer in the B16Braf V600E tumor model, 1x10 7 in vitro activated CD8 + T cells transduced with lentivectors were injected intravenously (i.v.) into recipient mice bearing 5 day-old tumors. For the OT-1 T cell transfer model, 2x10 6 OT-1 splenocytes or 10 6 OT-1 CD8 + T cells stimulated for 4-5 days in vitro were transferred i.v. into mice bearing 5 day-old B16 OVA tumors.

Method Details

Construction of Recombinant Adenoviral Vectors and Lentivectors Zhang and Ertl, 2014 Zhang Y.

Ertl H.C.J. The effect of adjuvanting cancer vaccines with herpes simplex virus glycoprotein D on melanoma-driven CD8+ T cell exhaustion. + T cell epitopes from human (h) or mouse (m)Trp-2, mTrp-1, hgp100 and mBraf V600E fused into herpes simplex virus (HSV) glycoprotein (g)D. We design the spacers between each epitopes using PAPROCI, Netchop 3.1 and IEBD Analysis Resource. Briefly, gDMelapoly or gDE7 sequences were inserted into the E1-deleted AdC68 viral molecular clone using I-CeuI and PI-SceI restriction enzyme sites. The constructed plasmids were used to transfect HEK 293 cells by calcium phosphate (Invitrogen). Cells infected with Ad vectors were harvest 7-10 days later upon plague formation. Virus was expanded on HEK 293 cells by serial infections and harvested by three cycles of freeze thawing. Virus was purified from cell-free supernatant after the third cycle of thawing by cesium chloride density ultracentrifugation. Molecular construction, rescue, purification and titration of the adenoviral vectors have been described (). The Melapoly sequence is composed of eight CD8T cell epitopes from human (h) or mouse (m)Trp-2, mTrp-1, hgp100 and mBraffused into herpes simplex virus (HSV) glycoprotein (g)D. We design the spacers between each epitopes using PAPROCI, Netchop 3.1 and IEBD Analysis Resource. Briefly, gDMelapoly or gDE7 sequences were inserted into the E1-deleted AdC68 viral molecular clone using I-CeuI and PI-SceI restriction enzyme sites. The constructed plasmids were used to transfect HEK 293 cells by calcium phosphate (Invitrogen). Cells infected with Ad vectors were harvest 7-10 days later upon plague formation. Virus was expanded on HEK 293 cells by serial infections and harvested by three cycles of freeze thawing. Virus was purified from cell-free supernatant after the third cycle of thawing by cesium chloride density ultracentrifugation. For production of lentivectors for HIF-1α KD, three pLKO.1 lentivectors containing shRNAs targeting different regions of HIF-1α or a pLKO.1 lentivector containing scrambled shRNA were obtained from the RNAi Consortium or Addgene. The selection marker Thy1.1 was cloned from the pLKO.3-Thy1.1 lentivector (Addgene) into each of the shRNA lentivectors. Lentivectors were generated using the 2nd generation lentivector package system (Addgene) by transfecting 293T cells with the packaging plasmid PsPAX2, the envelope plasmid PMD2.G and each of the shRNA-Thy1.1-expressing insert plasmids at a ratio of 3:1:1. Supernatants were collected 48 and 72 hr after transfection, spun at 3000rpm for 10 min and filtered through 0.45 μm filters. Lentivectors were concentrated by ultracentrifugation at 25,000 rpm, 4°C for 2 hr. Vector pellets were incubated with PBS at 4°C overnight before resuspension. Each lentivector, E1674, E1676 and E1677, was used to transduce activated CD8+ T cells independently for in vitro experiments, to control for potential off-target effect. E1676 was used in the vaccine model. A mixture of three lentivectors was used to transduce activated OT-1 cells in the OT-1 cell transfer model. For PD-L1 KD, four GIPZ lentiviral mouse CD274 shRNA constructs with TurboGFP and puromycin selection markers (Dharmacon) were used to produce lentivectors. A lentivector with scrambled non-silencing shRNA was used as control. The efficiency of PD-L1 KD was compared between different vectors. Two lentivectors shPDL1505-TurboGFP and shPDL1035-TurboGFP were independently used for downstream studies with B16Braf V600E cells; one was selected for studies with B16 OVA cells. Transduction with PD-L1 shRNA vectors or the control vector did not affect the growth of B16 OVA tumor cells in vitro.

In Vitro Stimulation of CD8+ T Cells and Drug Treatments Enriched CD8+ T cells from naive C57BL/6 mice were activated for 4 days in 6-well plates pre-coated with Ab to CD3 (5 μg/ml) and CD28 (1 μg/mL) (BD Bioscience). For some samples, cells were transferred for the last 16 hr to a hypoxia chamber. OT-1 splenocytes were stimulated with SIINFEKL peptide (2 μg/mL) and IL-2 (100 U/ml, Roche) for 48 hr. They were then maintained in medium containing IL-2 and split every other day. To study the impact of hypoxia on relatively resting CD8+ T cells, enriched CD8+ T cells were stimulated for 48 hr under normoxia. Cells were then removed from the plates, washed and replated in fresh medium with IL-2 (100 U/ml) for 96 hr, followed by culture in normoxia or hypoxia with IL-2 for another 36 hr before analysis. Cells were cultured in Roswell Park Memorial Institute (RPMI) medium without Glc (Life Technologies) supplemented with Glc (10 mM) or Gal (10 mM), 10% dialyzed FBS (Life Technologies), 20 mM HEPES, 2 mM Glutamax, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and 1% penicillin-streptomycin. Hypoxia experiments were performed in a Thermo Napco series 8000WJ CO 2 incubator equipped with nitrogen tank for O 2 replacement. O 2 level was kept at 1% during hypoxia experiments for the indicated time periods. In all assays cell viability was assessed before staining. Drugs and corresponding vehicle controls (all from Sigma) were added as follows: 2-deoxy-D-glucose (2-DG, 2 mM) or Fenofibrate (FF, 50 μM) for the entire culture period; Etomoxir (ETO, 200 μM) for the last 48 hr. FCCP (500 μM), Mito-TEMPO (500 μM) and oligomycin (1 mM) were used as positive and negative controls for mitochondrial stains. They were incubated with cells at 37°C for 20 min before samples were stained with DiOC6 and MitoSOX Red. DMSO concentrations were kept below 0.2% for all culture conditions.

Lentivector Transduction of CD8+ T Cells For in vitro experiments, 4x106 enriched CD8+ T cells were stimulated as described above for 24-28 hr. Freshly concentrated lentivectors were spun-inoculated into activated CD8+ T cells supplemented with polybrene (6 μg/ml, Santa Cruz) at 2000 rpm, 32°C for 2 hr. For tumor cell transduction, lentivectors were incubated with tumor cells in 1ml serum-free, antibiotic-free medium and spun at 2500 rpm, 30°C for 30 min. One ml of complete medium was added 6 hr later. Cells were washed 20 hr after transduction and stimulated for another 40 hr under normoxia or part-time hypoxia. For in vivo adoptive transfer experiment in the vaccine model, recipient mice had been challenged with tumor cells 5 days earlier and had been vaccinated with AdC68-gDMelapoly 2 days earlier. CD8+ T cells from spleens of naive C57Bl/6 mice were purified by negative selection using magnetic beads (MACS, STEMCELL Technologies). Enriched CD8+ T cells were stimulated with anti-CD3/CD28 Ab for 24 hr prior to lentivector transduction. Cells were washed 20 hr after transduction and cultured for an additional 48 hr in medium with IL-2 (100 U/ml) before transfer. For in vivo assays in the adoptive cell transfer model, OT-1 splenocytes were stimulated with peptide and IL-2 for 24 hr prior to spin inoculation with lentivectors. 72 hr later, dead cells were removed and transduced OT-1 CD8+ T cells were further purified by positive selection using CD90.1 microbeads (Miltenyi Biotec) before cell transfer.

Isolation of Lymphocytes from Mice Zhang and Ertl, 2014 Zhang Y.

Ertl H.C.J. The effect of adjuvanting cancer vaccines with herpes simplex virus glycoprotein D on melanoma-driven CD8+ T cell exhaustion. PBMCs and lymphocytes from spleens and lymph nodes were harvested as described (). Briefly, blood samples were collected by submandibular puncture and PBMCs were isolated by Histopaque (Sigma) gradient centrifugation. Single cell suspension was generated by mincing spleens and lymph nodes with mesh screens in Leibovitz’s L15 medium followed by passing cells through a 70 μm filter (Fisher Scientific). Red blood cells were lysed by 1x RBC lysis buffer (eBioscience). To obtain tumor-infiltrating lymphocytes, tumors were harvested, cut into small fragments and treated with 2 mg/ml Collagenase P, 1 mg/ml DNase I (all from Roche) and 2% FBS (Tissue Culture Biologicals) in Hank’s balanced salt solution (HBSS,1X, Thermo Fisher Scientific) under agitation for 1 hour. Tumor fragments were homogenized, filtrated through 70 μm strainers and lymphocytes were purified by Percoll-gradient centrifugation and washed with DMEM supplemented with 10% FBS. Pre-experiments were conducted to ensure that this treatment did not affect expression levels of any of the tested markers.

Ab, Staining and Flow Cytometry Cells were stained with a PE-labeled Trp-1-specific MHC class I (H-2Db) tetramer carrying the TAPDNLGYM peptide, an Alexa647-labeled HPV-16 E7-specific MHC class I (H-2Db) tetramer carrying the RAHYNIVTTF peptide or an Alexa647-labeled OVA-specific MHC class I (H-2Kb) tetramer carrying the SIINFEKL peptide (NIAID Tetramer Facility). Lymphocytes were stained with anti-CD8-PerCPCy5.5 or -Alexa700, CD4-PerCPCy5.5, CD44-PacBlue or PerCPCy5.5, LAG-3-APC or -PerCPCy5.5, PD-1-PE-Cy7 (clone RMP1-30) or -Brilliant violet (BV) 605 (clone 29F.1A12), CD127-BV421 or -Alexa700, CD62L-FTIC or BV785, KLRG1-PE-Cy7, CD45.1-Pacific Blue, Vα2-PE and Thy1.1-PerCPCy5.5 or -APC (all from Biolegend or eBioscience) and Amcyan fluorescent reactive dye (ThermoFisher). For human PBMC or TILs analysis, cells were stained with fluorochrome-labeled Ab to CD14/CD19 (dump gate), CD3, CD8, CD95, CD28 and CCR7 to identify subsets and with Ab to PD-1, PPAR-α, Cpt-1a and BODYPY FL C 6 . Levels of PD-1 and PD-L1 expression on tumor cells were determined with anti-PD-1-PE-Cy7 or anti-PD-L1-APC Ab used in comparison to isotype control Ab (Biolegend). For analysis of mitochondrial markers, cells were stained with MitoSOX Red (5μM, MROS) and DiOC6 (40nM, MMP) (ThermoFisher) at 37°C for 30 min under normoxia or hypoxia. For fatty acid uptake experiments, cells stimulated under different conditions in vitro or isolated from spleen and tumors of mice bearing day 14 or day 30 tumors were immediately incubated with 1 μM BODIPY FL C 16 (ThermoFisher) for 30 min at 37°C. Cells were washed twice with cold PBS before surface staining. For staining of T-bet, Eomes, total FoxO1, Cpt1a or PPAR-α cells were first stained for surface markers, then fixed and permeabilized with Foxp3/Transcription factor staining buffer and stained with T-bet-PE-Cy7, Eomes-Alexa488 (all from eBioscience), primary Ab against FoxO1 (C29H4, Cell Signaling Technology), anti-Cpt1a-Alexa 488 Ab or mouse IgG2b Isotype Ab; or anti-PPAR-α Ab (Abcam). Total FoxO1 was further determined by anti-rabbit secondary Ab staining (Cell Signaling Technology). PPAR-α was further detected with Streptavidin-BV605 (Biolegend). Phorphorylated (p)Akt was detected by staining cells with BD Phosflow buffers (Fix Buffer I and Perm Buffer III) and Phospho-Akt (Novus, clone 545007) Ab to Akt phosphorylated at serine 473. For HIF-1α and Glut staining, in ex vivo assays mice were perfused immediately after euthanasia with Hank’s balanced salt solution (HBSS) and heparin (10 units/ml) and then with 1 mM Cobalt (II) chloride (COCl 2 , Sigma-Aldrich) diluted in PBS. For both ex vivo and in vitro experiments, lymphocyte isolation and staining before fixation were performed in medium containing 200 μM COCl 2 . Cells were stained with Abs to cell surface markers for 30 min. Cells were fixed and permeabilized using the FoxP3 buffer kit, and stained for HIF-1α with anti-HIF-1α-Alexa700 Ab (R&D), or anti-Glut1-Alexa488 (Abcam) and the corresponding isotype control antibodies. For intracellular cytokine staining (ICS) of ex vivo stimulated lymphocytes, ∼106 cells per samples were cultured in DMEM containing 2% FBS and Golgiplug (Fisher Scientific, 1.5 μl/ml) for 6 hr with either a peptide pool (5 ug/ml for each peptide) including all CD8+ T cell epitopes expressed by gDMelapoly (mTrp-1 455-463 : TAPDNLGYA, mTrp-1 481-489 : IAVVAALLL, mTrp-2 522-529 : YAEDYEEL, hTp-2 180-188 : SVYDFFVWL, hTrp-2 343-357 : STFSFRNAL, mTrp-2 363-371 : SQVMNLHNL, hgp100 25-33 : KVPRNQDWL, mBraf 594-602 : FGLANEKSI); the E7 peptide: RAHYNIVTTF or SIINFEKL peptide (all from Genscript) or a rabies virus glycoprotein control peptide. For ICS performed with CD8+ T cells activated in vitro, ∼106 cells were transferred to 96 well plates in the original medium and stimulated with PMA (50 ng/ml), ionomycin (2 μg/ml) and Golgiplug for 4 hr under normoxia or hypoxia. Cells were stained with Ab to IFN-γ (APC or BV421), GrmB (APC, Life Technologies) and perforin (PE, eBioscience) after fixation/permeabilization (BD Pharmingen). Cells were analyzed by an LSRII (BD Biosciences). Data were analyzed with FlowJo (TreeStar).

T Cell Assays CD8+ T cells were analyzed by tetramer staining to assess phenotypes or by intracellular cytokine staining (ICS) following a 5-hour period of peptide stimulation in presence of GolgiPlug (BD Biosciences). The dominant CD8+ T response elicited by the AdC68-gDMelapoly vector is directed against the Trp-1 455 epitope (∼90% of MAA-specific CD8+ T cell response), which was assessed by tetramer staining. All peptides carrying the tumor antigen-specific epitopes of the vaccine inserts were used for ICS. For easier reference in the text we refer to both types of AdC68-gDMelapoly-induced CD8+ T cells as MAA-specific CD8+ T cells.

In Vivo Tumor Cell Survival Assay Mice were treated with isotype control- or α-PD-1 Ab (200 μg/mouse) 4 and 1 days before tumor cell challenge. Some of the mice had either been vaccinated with AdC68-gDMelapoly 14 days earlier, or been injected i.p. with in vitro activated OT-1 CD8+ T cells (105 cells/mouse) 4-6 hr before tumor cell challenge. PD-L1hi (scrambled shRNA lentivector transduced) or PD-L1lo (PD-L1 shRNA lentivectors transduced) B16Braf V600E tumor cells or B16 OVA cells were sorted based on lentivector GFP expression and passaged for several rounds in presence of puromycin to create transduced stable cell lines. Transduced tumor cells were labeled with high (2 μM) or low (0.2 μM) levels of CellTrace Violet (CTV), respectively, mixed at 1:1 ratio and injected i.p. into recipient mice at a dose of 106 cells/tumor cell subset. 1-day later mice were euthanized. Cells were collected by vigorously rinsing the peritoneal cavity with a trypsin solution. Cells were washed and stained with Ab to CD3, CD14 and CD19 as dump gates and with mAb to PD-L1. The relative recovery of CTVhiPD-L1hi vs. CTVloPD-L1lo cell subsets in each treatment groups was determined by flow cytometry.

BrdU Proliferation Assay Mice were injected i.p. with of BrdU (1.5-2 mg/mouse) and fed water-containing BrdU at a concentration of 0.8 mg/ml on days 9, 19 or 29 after vaccination; they were euthanized the next day and lymphocyte samples were analyzed for BrdU incorporation. Cells were first stained for surface markers and then for intracellular BrdU (1:50 dilution) according to the manufacture’s instruction (ThermoFisher).

Extracellular Flux Analysis and FAO Assay OCR and ECAR for CD8+ T cells stimulated under different conditions were measured with XF24 and XF96 Extracellular Flux Analyzers (Seahorse Bioscience). Hypoxia samples were prepared in a hypoxia chamber under 1% O 2 . Dead cells were removed by a dead cell removal kit (Miltenyi Biotec) and live cells were pre-incubated with 100 μM COCl 2 before being removed from the hypoxia chamber and entered into the Seahorse analyzer. To determine the contribution of FAO to OCR, 200 μM ETO was added 15 min before the Seahorse analysis. Briefly after repeated measures of basal respiration and lactate production, 1 μM OM was added to measure ATP leakage by OCR and glycolytic capacity by ECAR. 1.5 μM FCCP was then added to measure maximal respiration by OCR followed by addition of 100 nM Rotenone and 1 μM Antimycin A to determine spare respiratory capacity by OCR and then 100 mM 2-DG to determine glycolytic reserve by ECAR. For measuring oxidation of exogenous and endogenous FAs, cells activated in either Glc- or Gal-medium for 3 days were washed and transferred to substrate-limited Glc- or Gal-medium for overnight stimulation. Substrate limited media contained 0.5 mM Glc or Gal, 1 mM GlutaMAX, 0.5 mM carnitine (all form Sigma) and 1% dialyzed FBS. Samples were treated with either ETO or vehicle control 15 min before the assay. Palmitate: BSA or BSA was added just before the assay. The contributions of FAO to OCR was calculated as follows: Basal respiration due to exogenous FA oxidation= Basal Palm:BSA-ETO OCR rate – basal BSA-ETO OCR rate - OCR due to uncoupling by FFA; uncoupling by FFA= after OM injection, Palm:BSA-ETO OCR rate - BSA-ETO rate. Basal OCR due to endogenous FAs consumption = basal BSA-ETO OCR rate - basal BSA+ETO OCR rate.

Lipid and Glc Concentration Measurement in Tumor or Tissue Interstitial Fluid Wiig et al., 2003 Wiig H.

Aukland K.

Tenstad O. Isolation of interstitial fluid from rat mammary tumors by a centrifugation method. 13C 6 -Glc as the internal standard. Interstitial fluid from kidney, heart and mouse or human melanoma samples were collected by centrifugation as described () and snap frozen on dry ice. Mice were perfused with HBSS with 10 U/ml heparin before tissue collection. Concentrations of free FA species were determined by LC-MS. Absolute concentrations of Glc were measured by LC-MS upon adding-Glc as the internal standard.

Isotopic Labeling In Vitro For 13C 6 - Glc/Gal tracing in vitro, cells were cultured in Glc-free RPMI medium with 10 mM 13C 6 -Glc/Gal (Sigma or Cambridge) for 4 days. For 13C 16 -palmitate tracing in vitro, cells were stimulated for 3 days in Glc- or Gal-medium. On the night of day 3, some samples were transferred to 1% O 2 for overnight culture. 13C 16 -palmitate (Sigma) was first dissolved in 100% ethanol at 200 mM and conjugate to fatty acid-free BSA at a 5:1 molar ratio to a final concentration of 8 mM-13C 16 -palmitate-BSA by vortexing at 37°C for 3-4 hr with sonication. On day 4, samples were pelleted and replated in fresh medium with 10% delipidated FBS (Cocalico Biologicals) and 400 μM 13C 16 -palmitate-BSA. Hypoxia samples were returned to 1% O 2 . All samples were cultured for another 4 hr. Dead cells were removed. Samples were pelleted at 4000 rpm for 5 min. All collection procedures were conducted at 4°C. Cell numbers in each sample were determined. Metabolism was quenched and metabolites were extracted by adding 1 ml -80°C 80:20 methanol: water per million cells. After 20 min of incubation on dry ice, samples were centrifuged at 10000 g for 5 min. Insoluble pellets were re-extracted with 0.5 ml -80°C 80: 20 methanol: water on dry ice. The supernatants from two rounds of extraction were combined, dried under N 2 , resuspended in 100 μl water per million cells. Metabolites were normalized to cell numbers.

Isotope Labeling In Vivo Tumor-bearing mice were fasted for 16 hr. 13C 6 -Glc (Cambridge Isotope laboratories) diluted in PBS was given i.p. to mice at 2 g/kg. Spleens and tumors were collected 30 min later. 13C 16 -potassium palmitate (Sigma-Aldrich) was conjugated to FA-free BSA (6:1 molar ratio) and given to mice at ∼0.35 g/kg by oral gavage. 1 hour later 13C 16 -palmitate-BSA was given i.v. at 125 mg/kg. Spleens and tumors were collected 30 min later and cells were isolated on ice. To assess the potential effects of sample processing on lymphocyte metabolism, lymph nodes (LN) were snap frozen on dry ice immediately upon isolation. In the same experiments, single cell suspensions were prepared from other LNs of the same mice and incubated at 4°C for 2-2.5 hr prior to the metabolic analyses. CD44+CD8+ T cells from pooled spleen or tumor samples were stained and sorted at 4°C. Metabolites were extracted with -80°C 80: 20 methanol: water, dried under N 2 and resuspended in water at 100 mg tissue/ml or 106 cells/100 μl.

LC-MS Instrumentation and Method Development Lu et al., 2010 Lu W.

Clasquin M.F.

Melamud E.

Amador-Noguez D.

Caudy A.A.

Rabinowitz J.D. Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. 13C-abundance. Glycolytic and TCA metabolites were analyzed by reversed-phase ion-pairing chromatography coupled with negative-mode electrospray-ionization high-resolution MS on a stand-alone orbitrap (Thermo)(). Carnitine species were analyzed by reversed-phase ion pairing chromatography coupled with positive-mode electrospray-ionization on a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo); Liquid chromatography separation was achieved on a Poroshell 120 Bonus-RP column (2.1 mm ×150 mm, 2.7 μm particle size, Agilent). The total run time is 25 min, with a flow rate of 50 μl/min from 0 min to 12 min and 200 μl/min from 12 min to 25 min. Solvent A is 98: 2 water: acetonitrile with 10 mM amino acetate and 0.1% acetic acid; solvent B is acetonitrile. The gradient is 0-70% B in 12 min. All isotope-labeling patterns were corrected for naturalC-abundance.

Electron Microscopy CD44+CD8+ T cells were sorted from spleens and TILs of mice bearing day 30 tumors. They were fixed with 2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, overnight at 4°C. After washes in buffer, the samples were post-fixed in 2.0% osmium tetroxide for 1 hour at room temperature (RT), and then washed again in buffer followed by distilled H 2 O. After dehydration through a graded ethanol series, the samples were infiltrated and embedded in EMbed-812 (Electron Microscopy Sciences). Thin sections were stained with lead citrate and examined with a JEOL 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software.