2 nbd glucose

B16-F10 were obtained from ATCC. MC38 were a gift from Dario Vignali. B16^OVA (MO5) was obtained from Per Basse and Lou Falo. Both MC38 and B16^OVA have not been authenticated, but OVA expression was verified on B16^OVA by immunoblot and flow cytometry. Cell lines were obtained in 2014 and Mycoplasma testing was performed June 2014. Cell lines were not passaged more than 3 times before experimentation. Antibodies to CD8 (53-6.7), CD4 (GK1.5), PD-1 (29F.1812), Tim-3 (RMT3-23), CD44 (IM7), CD62L (MEL-14), TNFα (MP6-XT22), IFNγ (XMG1.2), CD11b (M1/70), CD11c (N4180), Ki67 (16A8), CD45 (30-F11), F4/80 (BM8), Ly6C (HK1.4), and propidium iodide were from BioLegend. Hypoxia staining was detected with an antibody to pimonidazole (Hypoxyprobe). Antibody to PD-1 (J43) and its hamster IgG control were obtained from Bio-X-Cell. CellTrace Violet was purchased from eBioscience. 2-NBD-glucose and metformin were purchased from Cayman Chemical. In all in vivo studies, mice were injected with 2.5 × 10⁵ tumor cells intradermally.

T cell metabolic output was measured by Seahorse technology as previously described (Scharping et al., 2016b ). Briefly, 100,000 T cells were seeded into Cell-Tak-coated XFe96 plates in minimal unbuffered assay media containing 25 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate. Cells received sequential injections of 2 μM oligmycin, 2 μM FCCP, 10 mM 2-deoxyglucose, and 0.5μM rotenone/antimycin A.
We assayed single-cell metabolic capacity by flow cytometry. Specifically, we utilized 2-NBD-glucose (Cayman Chemical) and MitoTracker FM dyes (ThermoFisher) to assay the propensity of cells to take up glucose or generate intermediates via their mitochondria. Nondraining and draining lymph node or tumor preparations were pulsed with 20 μM 2-NBDG in 5% FBS-containing media for 30 min at 37°0. Cells were surface stained and loaded with MitoTracker FM dyes to measure mitochondrial mass and function.

B16-F10 and LLC were obtained from ATCC. MC38 was obtained from Dario Vignali. B16^OVA (MO5) was obtained from Per Basse and Lou Falo. OVA-ex-pressing Vaccinia virus was originally generated by Yewdell and Bennink and obtained from Jonathan Powell. Most antibodies for flow cytometry were obtained from BioLegend. MitoTracker Green FM, MitoTracker Deep Red FM, tetramethylrhodamine ester (TMRE), and H2-DCFDA were obtained from ThermoFisher. VDAC antibody was obtained from Abcam. LC3B, pAktS473, pFoxo1/3a antibodies were obtained from Cell Signaling Technologies and detected after surface staining with simultaneous fixation and permabilization in 1.5% PFA madeup in 1X Permeabilization buffer (eBioscience). 2-NBD-glucose, m-divi-1, and Akt inhibitor VIII were purchased from Cayman Chemical. PGC1α antibody (H-300) was obtained from Santa Cruz Biotechnology, and was detected using the Foxp3 Fix/Perm kit (eBioscience) and Alexa Fluor 647 or Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G (IgG) (Jackson Immunoresearch). Anti-PD-1 blocking antibody (J43) and its hamster IgG control were obtained from Bio-X-Cell. CellTrace Violet was purchased from eBioscience, and CFSE was purchased from BioLegend.