Translating In Vitro T Cell Metabolic Findings to In Vivo Tumor Models of Nutrient Competition

Reductionist in vitro T cell assays have identified metabolic pathways critical for T cell function within the tumor microenvironment. We discuss the challenges of testing these concepts using in vivo tumor models. Reductionist in vitro T cell assays have identified metabolic pathways critical for T cell function within the tumor microenvironment. We discuss the challenges of testing these concepts using in vivo tumor models. Exciting new technologies and conceptual advancements have re-energized the study of the metabolic competition between tumors and the adaptive immune system (Chang et al., 2015Chang C.-H.H. Qiu J. O’Sullivan D. Buck M.D. Noguchi T. Curtis J.D. Chen Q. Gindin M. Gubin M.M. van der Windt G.J. et al.Metabolic competition in the tumor microenvironment is a driver of cancer progression.Cell. 2015; 162: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, Ho et al., 2015Ho P.-C.C. Bihuniak J.D. Macintyre A.N. Staron M. Liu X. Amezquita R. Tsui Y.-C.C. Cui G. Micevic G. Perales J.C. et al.Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses.Cell. 2015; 162: 1217-1228Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar, Sugiura and Rathmell, 2018Sugiura A. Rathmell J.C. Metabolic barriers to T cell function in tumors.J. Immunol. 2018; 200: 400-407Crossref PubMed Scopus (103) Google Scholar). Despite these advancements, there remains little discussion in the literature on how investigators should model nutrient depletion in the tumor microenvironment when studying immune cell/tumor cell metabolic interactions and competition. How can we more accurately distinguish and dissociate nutrient deprivation’s effects on immune cells from other immunosuppressive components of the tumor microenvironment in vivo? Are T cells engineered with altered metabolic programming specifically overcoming a nutrient limitation? Or are they simply better tumor-specific T cells that function better in all nutrient environments? This essay will lay out some of the critical issues facing the field and hopefully spur the development of improved approaches for testing new immune therapies. We believe that greater mechanistic understanding of immunometabolism will fully harness the clinical potential of immune therapies. In vivo, tumors alter the abundance of dozens of metabolites in the interstitial fluid relative to healthy tissue (Kamphorst et al., 2015Kamphorst J.J. Nofal M. Commisso C. Hackett S.R. Lu W. Grabocka E. Vander Heiden M.G. Miller G. Drebin J.A. Bar-Sagi D. et al.Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.Cancer Res. 2015; 75: 544-553Crossref PubMed Scopus (493) Google Scholar, Pavlova and Thompson, 2016Pavlova N.N. Thompson C.B. The emerging hallmarks of cancer metabolism.Cell Metab. 2016; 23: 27-47Abstract Full Text Full Text PDF PubMed Scopus (2918) Google Scholar). Most commonly solid tumor environments have been reported to be deprived of oxygen, glucose, glutamine, and multiple amino acids (e.g., arginine, tryptophan) and are highly acidic (Figure 1A). Different tumors and even separate regions of the same tumor can host diverse nutrient environments and can experience intermittent blood flow and nutrient supply (DeBerardinis and Chandel, 2016DeBerardinis R.J. Chandel N.S. Fundamentals of cancer metabolism.Sci. Adv. 2016; 2: e1600200Crossref PubMed Scopus (1482) Google Scholar, Vaupel et al., 1987Vaupel P. Fortmeyer H.P. Runkel S. Kallinowski F. Blood flow, oxygen consumption, and tissue oxygenation of human breast cancer xenografts in nude rats.Cancer Res. 1987; 47: 3496-3503PubMed Google Scholar). An abundance of pro-angiogenic factors (like vascular endothelial growth factor-A) promote disturbed vasculature characterized by large and leaky vessels, erratic branching, and irregular and slow blood flow, which inhibits efficient nutrient delivery (Carmeliet, 2005Carmeliet P. VEGF as a key mediator of angiogenesis in cancer.Oncology. 2005; 69: 4-10Crossref PubMed Scopus (1162) Google Scholar, Lanitis et al., 2015Lanitis E. Irving M. Coukos G. Targeting the tumor vasculature to enhance T cell activity.Curr. Opin. Immunol. 2015; 33: 55-63Crossref PubMed Scopus (189) Google Scholar). In addition, metabolites can be altered by elevated expression of enzymes expressed by tumor cells or tumor-associated antigen-presenting cells. For example, high expression of the enzyme indoleamine 2,3-dioxygenase in the tumor microenvironment can lead to local depletion of tryptophan and regulate T cell proliferation and induce apoptosis (Fallarino et al., 2002Fallarino F. Grohmann U. Vacca C. 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Zhang Y. Ron D. Mellor A.L. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase.Immunity. 2005; 22: 633-642Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar), highlighting that signaling pathways induced by low levels of nutrients can be as damaging to the anti-tumor response as the lack of nutrients to support immune cell function and growth. T cells require glucose, glutamine, and mitochondrial pathways for activation, maximal proliferation, and/or effector function (Cham and Gajewski, 2005Cham C.M. Gajewski T.F. Glucose availability regulates IFN-gamma production and p70S6 kinase activation in CD8+ effector T cells.J. Immunol. 2005; 174: 4670-4677Crossref PubMed Scopus (236) Google Scholar, Macintyre et al., 2014Macintyre A.N. Gerriets V.A. Nichols A.G. Michalek R.D. Rudolph M.C. Deoliveira D. Anderson S.M. Abel E.D. Chen B.J. Hale L.P. Rathmell J.C. The glucose transporter Glut1 is selectively essential for CD4 T cell activation and effector function.Cell Metab. 2014; 20: 61-72Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, Procaccini et al., 2016Procaccini C. Carbone F. Di Silvestre D. Brambilla F. De Rosa V. Galgani M. Faicchia D. Marone G. Tramontano D. Corona M. et al.The proteomic landscape of human ex vivo regulatory and conventional T cells reveals specific metabolic requirements.Immunity. 2016; 44: 406-421Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Ron-Harel et al., 2016Ron-Harel N. Santos D. Ghergurovich J.M. Sage P.T. Reddy A. Lovitch S.B. Dephoure N. Satterstrom F.K. Sheffer M. Spinelli J.B. et al.Mitochondrial biogenesis and proteome remodeling promote one-carbon metabolism for T cell activation.Cell Metab. 2016; 24: 104-117Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, Sena et al., 2013Sena L.A. Li S. Jairaman A. Prakriya M. Ezponda T. Hildeman D.A. Wang C.-R.R. Schumacker P.T. Licht J.D. Perlman H. et al.Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling.Immunity. 2013; 38: 225-236Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Aerobic glycolysis serves the increased biosynthetic demands of highly proliferating cells. Glycolytic intermediates can produce nucleotides, lipids, and amino acids necessary for cellular proliferation in both T cells and cancer cells (Vander Heiden et al., 2009Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (10144) Google Scholar). Similar reliance on aerobic glycolysis between proliferating cancer cells and activated T cells intensifies a competition for limited nutrients within the tumor microenvironment (Frauwirth et al., 2002Frauwirth K.A. Riley J.L. Harris M.H. Parry R.V. Rathmell J.C. Plas D.R. Elstrom R.L. June C.H. Thompson C.B. The CD28 signaling pathway regulates glucose metabolism.Immunity. 2002; 16: 769-777Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar). Competition for glucose also plays a clear role in limiting effective anti-tumor responses in vivo (Chang et al., 2015Chang C.-H.H. Qiu J. O’Sullivan D. Buck M.D. Noguchi T. Curtis J.D. Chen Q. Gindin M. Gubin M.M. van der Windt G.J. et al.Metabolic competition in the tumor microenvironment is a driver of cancer progression.Cell. 2015; 162: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, Ho et al., 2015Ho P.-C.C. Bihuniak J.D. Macintyre A.N. Staron M. Liu X. Amezquita R. Tsui Y.-C.C. Cui G. Micevic G. Perales J.C. et al.Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses.Cell. 2015; 162: 1217-1228Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar). Aerobic glycolysis is required for maximal IFN-γ production by effector T cells though at least two independent mechanisms. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can inhibit translation of IFN-γ mRNA, while another study has demonstrated that the glycolytic enzyme, lactate dehydrogenase A (LDHA), promotes histone acetylation along the IFN-γ locus (Chang et al., 2013Chang C.-H.H. Curtis J.D. Maggi Jr., L.B. Faubert B. Villarino A.V. O’Sullivan D. Huang S.C. van der Windt G.J. Blagih J. Qiu J. et al.Posttranscriptional control of T cell effector function by aerobic glycolysis.Cell. 2013; 153: 1239-1251Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, Peng et al., 2016Peng M. Yin N. Chhangawala S. Xu K. Leslie C.S. Li M.O. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism.Science. 2016; 354: 481-484Crossref PubMed Scopus (423) Google Scholar). Although some attempts to increase nutrient availability have been successful, we have little understanding of how to make T cells fit better in a nutrient-limiting environment (Chang et al., 2015Chang C.-H.H. Qiu J. O’Sullivan D. Buck M.D. Noguchi T. Curtis J.D. Chen Q. Gindin M. Gubin M.M. van der Windt G.J. et al.Metabolic competition in the tumor microenvironment is a driver of cancer progression.Cell. 2015; 162: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, Lanitis et al., 2015Lanitis E. Irving M. Coukos G. Targeting the tumor vasculature to enhance T cell activity.Curr. Opin. Immunol. 2015; 33: 55-63Crossref PubMed Scopus (189) Google Scholar). Due to dysfunctional vasculture and the high glycolytic rate of many tumor cells, the tumor microenvrionment is often acidic because of high lactate concentrations (Figure 1B). Both tumor and T cells rely on monocarboxylate transporters to secrete and uptake lactate generated from glycolysis and pyruvate. High lactate concentrations in the environment prevent T cells from efficiently transporting lactate out of the cell and increase intracellular acidity and inhibit functionality and NFAT activity (Brand et al., 2016Brand A. Singer K. Koehl G.E. Kolitzus M. Schoenhammer G. Thiel A. Matos C. Bruss C. Klobuch S. Peter K. et al.LDHA-associated lactic acid production blunts tumor immunosurveillance by T and NK cells.Cell Metab. 2016; 24: 657-671Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar, Fischer et al., 2007Fischer K. Hoffmann P. Voelkl S. Meidenbauer N. Ammer J. Edinger M. Gottfried E. Schwarz S. Rothe G. Hoves S. et al.Inhibitory effect of tumor cell-derived lactic acid on human T cells.Blood. 2007; 109: 3812-3819Crossref PubMed Scopus (1086) Google Scholar). Brand et al. demonstrated that melanoma tumor cells engineered to have low lactate dehydrogenase A (LDHA) activity have greater immune infiltration and activity than tumor cells with normal LDHA expresion. LDHA is the glycolytic enzyme required for conversion of pyruvate to lactate. Furthermore, the administration of the proton pump inhibitor esomeprazole can normalize tumor pH in vivo and when combined with immunotherapy can promote tumor clearance (Calcinotto et al., 2012Calcinotto A. Filipazzi P. Grioni M. Iero M. De Milito A. Ricupito A. Cova A. Canese R. Jachetti E. Rossetti M. et al.Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes.Cancer Res. 2012; 72: 2746-2756Crossref PubMed Scopus (387) Google Scholar). While glucose deprivation and high lactate concentrations inhibit anti-tumor responses, they also promote immunosuppressive T regulatory cell and macrophage polarization and function (Angelin et al., 2017Angelin A. Gil-de-Gómez L. Dahiya S. Jiao J. Guo L. Levine M.H. Wang Z. Quinn 3rd, W.J. Kopinski P.K. Wang L. et al.Foxp3 reprograms T cell metabolism to function in low-glucose, high-lactate environments.Cell Metab. 2017; 25: 1282-1293Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, Colegio et al., 2014Colegio O.R. Chu N.-Q.Q. Szabo A.L. Chu T. Rhebergen A.M. Jairam V. Cyrus N. Brokowski C.E. Eisenbarth S.C. Phillips G.M. et al.Functional polarization of tumour-associated macrophages by tumour-derived lactic acid.Nature. 2014; 513: 559-563Crossref PubMed Scopus (1511) Google Scholar). As T cells become exposed to chronic antigen in the tumor microenvironment, they begin to terminally differentiate and become exhausted. As exhaustion becomes more severe, T cells increase expression of multiple inhibitory receptors, while simultaneously inhibiting costimulatory receptor expression (Figure 1C). Inhibitory receptors can inhibit co-stimulation and phosphorylation events downstream of T cell receptor (TCR) activation and prevent activation signals (Akt and mTORC1) necessary for increasing surface expression of the major glucose transporter, Glut-1, in T cells (Jacobs et al., 2008Jacobs S.R. Herman C.E. Maciver N.J. Wofford J.A. Wieman H.L. Hammen J.J. Rathmell J.C. Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways.J. Immunol. 2008; 180: 4476-4486Crossref PubMed Scopus (553) Google Scholar, Parry et al., 2005Parry R.V. Chemnitz J.M. Frauwirth K.A. Lanfranco A.R. Braunstein I. Kobayashi S.V. Linsley P.S. Thompson C.B. Riley J.L. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms.Mol. Cell. Biol. 2005; 25: 9543-9553Crossref PubMed Scopus (1317) Google Scholar, Siska et al., 2016Siska P.J. van der Windt G.J. Kishton R.J. Cohen S. Eisner W. MacIver N.J. Kater A.P. Weinberg J.B. Rathmell J.C. Suppression of Glut1 and glucose metabolism by decreased Akt/mTORC1 signaling drives T cell impairment in B cell leukemia.J. Immunol. 2016; 197: 2532-2540Crossref PubMed Scopus (85) Google Scholar). Without sufficient surface expression of Glut-1, the activated T cells are unable to properly upregulate glycolysis and have inhibited proliferation and effector function. Upregulation of mitochondrial pathways (oxidative phosphorylation and one-carbon metabolism) is essential for proper T cell proliferation. (Chang et al., 2013Chang C.-H.H. Curtis J.D. Maggi Jr., L.B. Faubert B. Villarino A.V. O’Sullivan D. Huang S.C. van der Windt G.J. Blagih J. Qiu J. et al.Posttranscriptional control of T cell effector function by aerobic glycolysis.Cell. 2013; 153: 1239-1251Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, Procaccini et al., 2016Procaccini C. Carbone F. Di Silvestre D. Brambilla F. De Rosa V. Galgani M. Faicchia D. Marone G. Tramontano D. Corona M. et al.The proteomic landscape of human ex vivo regulatory and conventional T cells reveals specific metabolic requirements.Immunity. 2016; 44: 406-421Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Ron-Harel et al., 2016Ron-Harel N. Santos D. Ghergurovich J.M. Sage P.T. Reddy A. Lovitch S.B. Dephoure N. Satterstrom F.K. Sheffer M. Spinelli J.B. et al.Mitochondrial biogenesis and proteome remodeling promote one-carbon metabolism for T cell activation.Cell Metab. 2016; 24: 104-117Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, Ron-Harel et al., 2015Ron-Harel N. Sharpe A.H. Haigis M.C. Mitochondrial metabolism in T cell activation and senescence: a mini-review.Gerontology. 2015; 61: 131-138Crossref PubMed Scopus (35) Google Scholar). In addition, mitochondria play roles beyond producing energy and biosynthesis, though epigenetic and signaling roles during T cell activation (Minocherhomji et al., 2012Minocherhomji S. Tollefsbol T.O. Singh K.K. Mitochondrial regulation of epigenetics and its role in human diseases.Epigenetics. 2012; 7: 326-334Crossref PubMed Scopus (114) Google Scholar, Sena et al., 2013Sena L.A. Li S. Jairaman A. Prakriya M. Ezponda T. Hildeman D.A. Wang C.-R.R. Schumacker P.T. Licht J.D. Perlman H. et al.Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling.Immunity. 2013; 38: 225-236Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Chronic antigen exposure affects metabolic pathways beyond glycolysis, by inhibiting mitochondrial functions (Figure 1D). Exhausted T cells in models of cancer or chronic virus infection often have defects in mitochondria number, size, and voltage potential and function (Bengsch et al., 2016Bengsch B. Johnson A.L. Kurachi M. Odorizzi P.M. Pauken K.E. Attanasio J. Stelekati E. McLane L.M. Paley M.A. Delgoffe G.M. Wherry E.J. 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One study has proposed that the tumor microenvironment downregulates mitochondrial biosynthesis by inhibiting expression of PPAR-gamma coactivator 1α (PGC1α), while another has suggested ROS-induced damage may induce decreased mitochondrial mass (Scharping et al., 2016Scharping N.E. Menk A.V. Moreci R.S. Whetstone R.D. Dadey R.E. Watkins S.C. Ferris R.L. Delgoffe G.M. The tumor microenvironment represses T cell mitochondrial biogenesis to drive intratumoral T cell metabolic insufficiency and dysfunction.Immunity. 2016; 45: 374-388Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, Siska et al., 2017Siska P.J. Beckermann K.E. Mason F.M. Andrejeva G. Greenplate A.R. Sendor A.B. Chiang Y.J. Corona A.L. Gemta L.F. Vincent B.G. et al.Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma.JCI Insight. 2017; 2: 93411Crossref PubMed Scopus (176) Google Scholar). Scharping et al. have successfully demonstrated that overexpression of PGC1α in tumor-specific T cells is able to prevent downregulation of mitochondrial mass and enhance T cell functionality in the tumor microenvironment. Tumor-infiltrating lymphocytes are exposed to large amounts of reactive oxygen species (ROS, Figure 1E). ROS are highly chemically reactive and can damage cellular structures and inhibit T cell activation. Production of ROS drastically increases in T cells exposed to hypoxia and is one of the major mechanisms of immune suppression by myeloid-derived suppressor cells (Kusmartsev et al., 2004Kusmartsev S. Nefedova Y. Yoder D. Gabrilovich D.I. Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species.J. Immunol. 2004; 172: 989-999Crossref PubMed Scopus (658) Google Scholar, Tafani et al., 2016Tafani M. Sansone L. Limana F. Arcangeli T. De Santis E. Polese M. Fini M. Russo M.A. The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression.Oxid. Med. Cell. Longev. 2016; 2016: 3907147Crossref PubMed Scopus (205) Google Scholar). Myeloid-derived suppressor cells are a heterogeneous immunosuppressive subset of cells overexpressed in patients and mice with cancer. While ROS are necessary for IL-2 production and proliferation by T cells, it remains unclear the quantities in which ROS will become harmful to T cells (Sena et al., 2013Sena L.A. Li S. Jairaman A. Prakriya M. Ezponda T. Hildeman D.A. Wang C.-R.R. Schumacker P.T. Licht J.D. Perlman H. et al.Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling.Immunity. 2013; 38: 225-236Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Exposure to oxidative stress by low doses of exogenous hydrogen peroxide is sufficient to inhibit T cell functionality and survival (Ligtenberg et al., 2016Ligtenberg M.A. Mougiakakos D. Mukhopadhyay M. Witt K. Lladser A. Chmielewski M. Riet T. Abken H. Kiessling R. Coexpressed catalase protects chimeric antigen receptor-redirected T cells as well as bystander cells from oxidative stress-induced loss of antitumor activity.J. Immunol. 2016; 196: 759-766Crossref PubMed Scopus (120) Google Scholar). Given the metabolic challenges tumor-specific T cells face, there have been numerous attempts to mitigate these effects via pharmacological or genetic modifications to improve tumor-specific T cell therapy in vivo. However, our interpretation of in vivo studies has been hindered by numerous technical and biological challenges. It is incredibly difficult to distinguish whether newly designed immune interventions targeting the metabolic challenges of solid tumors have generated T cells that operate better in nutrient depleted environments or simply have made a better T cell (that operates better in all environments). The key to distinguishing these possibilities are (1) identifying how a modification improves the anti-tumor response and (2) understanding how individual metabolic interactions lead to distinct functional outcomes. For example, enhancing mitochondrial fusion, increasing L-arginine media concentrations, and inhibiting glycolytic metabolism while T cells are being expanded for adoptive cell therapy all enhance the therapeutic activity of the infused T cells (Buck et al., 2016Buck M.D. O’Sullivan D. Klein Geltink R.I. Curtis J.D. Chang C.-H.H. Sanin D.E. Qiu J. Kretz O. Braas D. van der Windt G.J. et al.Mitochondrial dynamics controls t cell fate through metabolic programming.Cell. 2016; 166: 63-76Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, Geiger et al., 2016Geiger R. Rieckmann J.C. Wolf T. Basso C. Feng Y. Fuhrer T. Kogadeeva M. Picotti P. Meissner F. Mann M. et al.L-arginine modulates T cell metabolism and enhances survival and anti-tumor activity.Cell. 2016; 167: 829-842Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, Sukumar et al., 2013Sukumar M. Liu J. Ji Y. Subramanian M. Crompton J.G. Yu Z. Roychoudhuri R. Palmer D.C. Muranski P. Karoly E.D. et al.Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function.J. Clin. Invest. 2013; 123: 4479-4488Crossref PubMed Scopus (565) Google Scholar). All of these interventions alter T cell metabolism, but do they enable T cells to function better in the tumor microenvironment? It is challenging to envision a mechanism by which temporarily expanding T cells in low glucose empowers them to function better in the tumor microenvironment. Rather, previous studies demonstrate that T cells expanded in low glucose retain a less differentiated phenotype, which enables improved engraftment of the expanded T cells, which correlates with improved tumor control (Gattinoni et al., 2011Gattinoni L. Lugli E. Ji Y. Pos Z. Paulos C.M. Quigley M.F.F. Almeida J.R. Gostick E. Yu Z. Carpenito C. et al.A human memory T cell subset with stem cell-like properties.Nat. Med. 2011; 17: 1290-1297Crossref PubMed Scopus (1234) Google Scholar). Expansion in the presence of higher levels of L-arginine also results in T cells that are less differentiated and more reliant on oxidative phosphorylation instead of glycolysis (Geiger et al., 2016Geiger R. Rieckmann J.C. Wolf T. Basso C. Feng Y. Fuhrer T. Kogadeeva M. Picotti P. Meissner F. Mann M. et al.L-arginine modulates T cell metabolism and enhances survival and anti-tumor activity.Cell. 2016; 167: 829-842Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar). These cells have higher intracellular concentrations of L-arginine, which may make them resistant to low arginine levels in the tumor microenvironment. Likewise, T cells with enhanced mitochondrial fusion are less differentiated in culture and exhibit greater reliance on oxidative phosphorylation (Buck et al., 2016Buck M.D. O’Sullivan D. Klein Geltink R.I. Curtis J.D. Chang C.-H.H. Sanin D.E. Qiu J. Kretz O. Braas D. van der Windt G.J. et al.Mitochondrial dynamics controls t cell fate through metabolic programming.Cell. 2016; 166: 63-76Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). Enhanced fusion may enhance mitochondrial function and combat mitophagy frequently observed during nutrient deprivation (Rambold et al., 2011Rambold A.S. Kostelecky B. Elia N. Lippincott-Schwartz J. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation.Proc. Natl. Acad. Sci. USA. 2011; 108: 10190-10195Crossref PubMed Scopus (758) Google Scholar). How do we determine to what extent differentiation or improved fitness in the tumor microenvironment results in improved tumor clearance? Furthermore, any attempts to modulate metabolism in the tumor microenvironment is likely going to cascade and affect many of the surrounding cells and may have cell-specific effects. For example, enhancing tumor glycolysis is sufficient to decrease available glucose for T cells but may also create a better environment for tumor-associated macrophages and T regulatory cells (Angelin et al., 2017Angelin A. Gil-de-Gómez L. Dahiya S. Jiao J. Guo L. Levine M.H. Wang Z. Quinn 3rd, W.J. Kopinski P.K. Wang L. et al.Foxp3 reprograms T cell metabolism to function in low-glucose, high-lactate environments.Cell Metab. 2017; 25: 1282-1293Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, Chang et al., 2015Chang C.-H.H. Qiu J. O’Sullivan D. Buck M.D. Noguchi T. Curtis J.D. Chen Q. Gindin M. Gubin M.M. van der Windt G.J. et al.Metabolic competition in the tumor microenvironment is a driver of cancer progression.Cell. 2015; 162: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, Netea-Maier et al., 2018Netea-Maier R.T. Smit J.W.A. Netea M.G. Metabolic changes in tumor cells and tumor-associated macrophages: A mutual relationship.Cancer Lett. 2018; 413: 102-109Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In addition, simply increasing T cell activity is sufficient to modulate metabolites in the serum of mice, and the effects of these changes on other cell types are not understood (Miyajima et al., 2017Miyajima M. Zhang B. Sugiura Y. Sonomura K. Guerrini M.M. Tsutsui Y. Maruya M. Vogelzang A. Chamoto K. Honda K. et al.Metabolic shift induced by systemic activation of T cells in PD-1-deficient mice perturbs brain monoamines and emotional behavior.Nat. Immunol. 2017; 18: 1342-1352Crossref PubMed Scopus (72) Google Scholar). Future studies must work to better distinguish mechanisms of metabolic immune interventions to provide mechanistic understanding of immunometabolism (Figure 2A). Unlike standard epigenetic or transcriptional changes that often occur over several hours or days, many metabolic changes occur incredibly quickly in response to different environments. This may even be more pronounced in immune cells, because these cells readily circulate and traffic to diverse areas of the body, and thus must be able to adapt rapidly in diverse metabolic environments. While researchers can observe cell-intrinsic differences of different T cell subsets in vitro, how much information is lost because of isolation and prolonged in vitro expansion (Dimeloe et al., 2016Dimeloe S. Mehling M. Frick C. Loeliger J. Bantug G.R. Sauder U. Fischer M. Belle R. Develioglu L. Tay S. et al.The immune-metabolic basis of effector memory CD4+ T cell function under hypoxic conditions.J. Immunol. 2016; 196: 106-114Crossref PubMed Scopus (55) Google Scholar, Pan et al., 2017Pan Y. Tian T. Park C.O. Lofftus S.Y. Mei S. Liu X. Luo C. O’Malley J.T. Gehad A. Teague J.E. et al.Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism.Nature. 2017; 543: 252-256Crossref PubMed Scopus (376) Google Scholar, Procaccini et al., 2016Procaccini C. Carbone F. Di Silvestre D. Brambilla F. De Rosa V. Galgani M. Faicchia D. Marone G. Tramontano D.