Targeting immuno-metabolism to improve anti-cancer therapies
Introduction
Many high-quality reviews on T cell metabolism have been published recently and the reader is encouraged to seek these out [1], [2]. In the classic paradigm, quiescent T cells rely on oxidative metabolism, where nutrients are transported into the mitochondria, fats are oxidized through fatty acid oxidation (FAO), intermediates are shunted into the Tricarboxylic acid cycle (TCA) to generate FADH2 and NADH, and these reducing agents are used by the electron transport chain to generate ATP through oxidative phosphorylation (OXPHOS). When T cells become activated, signaling through the TCR and the increased metabolic demands of a rapid division, drive an increase in the rate of glycolysis, with active conversion of pyruvate to lactate instead of being shunted into the TCA cycle [3], [4]. This increase in glycolysis, despite the presence of sufficient oxygen, is often referred to as the “Warburg effect”, eponymously named for Otto Warburg's seminal discovery of a similar aerobic glycolysis described in cancer cells at the beginning of the last century [5]. During resolution of an immune response, surviving T cells convert to memory T cells and again become reliant on oxidative metabolism [6], [7], [8], [9].
Until recently, little was known about the process of lipid transport and lipolysis in T cells. That is beginning to change and the first of the new studies suggests that lipids can be generated de-novo inside of cells, rather than be transported from outside, followed by breakdown of lipid by intracellular lipases including lysosomal acid lipase (LAL) [10]. More recently, this viewpoint has expanded to demonstrate that both lipid uptake and synthesis are important for robust T cell proliferation following antigen recognition. Specifically, the mTORC1-PPARγ pathway was found to be critical to drive fatty acid uptake in activated CD4+ T cells and this adaptation was absolutely necessary to achieve complete activation and rapid proliferation of both naive and memory CD4+ cells [11]. In addition, uptake of free fatty acids (FFAs) by fatty acid binding protein 4 and 5 (FABP4/FABP5) was determined to be critical for optimal performance of tissue resident memory T cells, and genetic knockdown of these vital proteins yielded T cells with poor protection against viral skin infections [12].
To generate energy from fat oxidation, cytosolic FFAs are conjugated to an acyl group by coenzyme A, chaperoned to the mitochondria, and the CoA moiety is replaced with carnitine by the molecule carnitine palmitoyl transferase 1 alpha (CPT1α). This acyl-carnitine species is then transported across the mitochondrial membrane by carnitine translocase, followed by deconjugation of carnitine by CPT2, which converts acylcarnitines back to a long-chain acyl-CoA molecules. Intramitochondrial Acyl-CoA moieties then become available for catabolism through the process of β-oxidation [13]. The end-product of FAO is Acetyl-CoA, which when shuttled into the TCA cycle, produces the reducing equivalents NADH and FADH2 which can then be utilized by the electron transport chain to produce ATP through OXPHOS. Inhibition of CPT1α by etomoxir has been shown to significantly impact the survival of regulatory T cells (Treg) [14], leading to speculation that FAO is required for Treg maintenance and generation. However, etomoxir can have off target effects unrelated to fat oxidation [15], and most of the studies on Treg and FAO analyzed Treg generation following prolonged in vitro culture. Furthermore, inhibition of fat oxidation did not block human inducible Treg generation [16], suggesting that the full impact of fat oxidation on Treg development and function await further investigation.
Regulation of enzymes and metabolites in both the TCA and FAO pathways are critically important to understanding T cell metabolism, and the reader is encouraged to seek out multiple detailed reviews published recently on this subject [32], [33], [34]. To briefly summarize the TCA cycle and its enzymes, acetyl-CoA, generated by either FAO or glycolysis, is joined to oxaloacetate by citrate synthase to form citrate. Citrate is then converted to isocitrate by aconitase, which is further processed to α-ketoglutarate by isocitrate-dehydrogenase (IDH). Processing of a-ketoglutarate by a-ketoglutarate dehydrogenase to form succinyl-CoA is followed by formation of succinate by succinate thiokinase. Succinate is reduced by succinate dehydrogenase to fumarate which is processed by fumarase to form l-malate. Finally, l-malate is reduced by malate dehydrogenase (MDH) to form oxaloacetate, completing the cycle.
To date, little work has analyzed the effect of specific TCA cycle enzyme inhibition on T cell proliferation and function. However, recently LW6, a putative HIF-1α inhibitor, was shown to specifically target malate dehydrogenase-2 (MDH2), blocking the oxidation of malate and reducing NADH and FADH2 generation [17]. LW6 was then used to interrogate the effect of MDH2 inhibition on T cell proliferation and apoptosis. Blockade of MDH2 in vitro reduced T cell proliferation, decreased apoptosis, and mediated metabolic adaptations to compensate for increased energy loss [18]. Another TCA cycle enzyme linked to T cells is isocitrate dehydrogenase 2 (IDH2). Mutations in IDH2 are found in angioimmunoblastic T cell lymphoma, where mutated IDH2 catalyzes transformation of isocitrate to 2-hydroxyglutarate, an oncogenic metabolite that alters histone methylation [19], in a process that is similar to what is observed in some forms of acute myelogenous leukemia. Future insights into the role of TCA cycle enzymes and T cell function may result from detailed observation of individuals with germline mutations in genes such as fumarase [20] and succinate dehydrogenase [21], or from the study of T cell function following exposure to specific enzyme inhibitors.
As previously described, activated T cells undergo rapid metabolic reprogramming that engages both glycolytic and oxidative metabolism. Adoption of these specific metabolic pathways is controlled by a rapid increase in the expression of transcription factors which drive bioenergetic programs. One such transcription factor is the proto-oncogene Myc, which is known to upregulate a host of genes involved in glycolysis [24]. Activation of Myc, in response to TCR signaling, has been understood for many years to be responsible for T cell proliferation [22], [23], and Myc was recently shown to be responsible for promoting both glycolysis and glutaminolysis in activated T cells [25]. HIF-1α, another transcription factor critical is heavily involved with T cell development, proliferation, and differentiation [27], also drives T cell glycolysis [26].
To upregulate oxidative metabolism, immune cells utilize several transcription factors including peroxisome proliferator activated receptor (PPAR) family and its co-factors (e.g. PPARγ co-activator 1-α) and PPAR family members exert significant effects on T cell development and function [29], activities likely married to their metabolic regulation. For example, PPAR-α drives transcription of genes in FAO pathways including CPT1α, fatty acid binding protein 4 (FABP4) and FABP5 [28]. Sterol regulatory element binding proteins (SREBP) are additional transcription factors that control lipid metabolism, often by influencing cholesterol handling and regulating control of FAO [30]. SREBPs reprogram lipid metabolism during T cell activation, and aid in the clonal expansion of cells in response to antigenic stimuli, but do not participate in homeostatic proliferation [31]. That said, there are few studies directly linking changes in lipid metabolism to anti-cancer responses, and when lipid metabolism is involved in T cell responses, e.g. following allogeneic HSCT, we will highlight these findings in greater detail. For the remainder of this review, when oxidative metabolism is referred to, it will conceptually include any lipid metabolism that generates ATP through the TCA cycle and/or subsequent OXPHOS.
While the classic paradigm of activated T cells utilizing glycolysis provides a conceptual framework for understanding T cell metabolism, significant caveats exist. Many of the classic experiments on immunometabolism were performed on cells cultured and activated in vitro, where bioenergetic substrates such as glucose, glutamine, and oxygen are supplied in extra-physiologic amounts [27], [35]. In addition, metabolic pathways do not always represent and “either/or” scenario and T cells can upregulate both aerobic glycolysis and oxidative metabolism simultaneously, following either in vitro and in vivo activation. There is speculation that these processes serve distinct purposes, for example with OXPHOS necessary for early phase proliferation, while glycolysis supports T cell effector functions, specifically interferon gamma production [36]. Similar findings exist in alloreactive T cells during graft-versus-host disease, where activated cells rapidly acidify their media ex vivo, but also robustly increase their oxygen consumption rates (personal observation). Finally, proteins like AMP-activated protein kinase (AMPK), a cellular energy sensor known to drive fat oxidation in multiple systems, is upregulated in effector T cells [37], where the classic paradigm suggests glycolysis would predominate. These findings all suggest that metabolic adoption of glycolysis versus oxidative metabolism is not an “all or none” event and that metabolic reprogramming is likely more nuanced and context dependent than is commonly assumed.
It should be mentioned that metabolic substrates beyond glucose, oxygen and fatty acids are also required during T cell proliferation and function. Amino acids, specifically, have been studied for their role in promoting essential T cell functions and this data is reviewed nicely elsewhere [38]. Briefly, glutamine, arginine, and leucine are all required during activation and early effector function. T cells cultured ex vivo are highly sensitive to the glutamine concentration of the media and T cell receptor (TCR) activation leads to an upregulation of glutamine transporters and increase in glutamine import [39]. This increased uptake is regulated, at least in part, by the transport protein Slc1a5, as absence of Slc1a5 decreases glutamine transport and impairs T helper (Th) 1 and Th17 responses [40]. T cell activation also causes large decreases in intracellular l-arginine levels, and supplementation of the media with excess arginine increases T cell survival and enhanced cytotoxicity against tumor cells in vivo [41]. Finally, the role of leucine in T cells was elucidated through study of the neutral amino acid transporter Slc7a5. During activation, T cells upregulate expression of Slc7a5 and absence of this transporter renders cells unable to clonally expand, metabolically reprogram, or differentiate in response to antigen, findings that were later shown to a be a result of poor leucine uptake [42].
The remainder of this review will step away from the broader aspects of T cell metabolism and instead focus on the metabolic pathways present in T cells during their interactions with various cancers and tumor microenvironments. Both positive and negative effects of modulating T cell metabolism in these contexts will be considered and speculation given on ways that targeting metabolic pathways might improve cancer treatments and overall clinical outcomes.
Section snippets
Allogeneic T cells activate mitochondrial metabolism
Hematopoietic stem cell transplantation (HSCT) from an allogeneic donor is a curative treatment for leukemia, lymphoma, and related hematologic malignancies. During this procedure, the patient's native immune system is ablated by chemotherapy and/or irradiation, followed by infusion of donor bone marrow or peripheral blood cells to facilitate immune reconstitution [43]. The major side-effect of allogeneic HSCT is graft-versus-host disease (GVHD), where alloreactive T cells from the graft
Leveraging metabolism to enhance T cell responses: CAR T cells
In the previous section, the discussion focused on modulating T cell metabolism to limit pathogenic immune responses. In other contexts, it may be advantageous to maximize T cell responses, as in the case of chimeric antigen receptor (CAR) T cells responding against target antigens on cancer cells [9]. In their simplest form, CARs consist of an extracellular antigen-recognition domain (commonly an antibody single-chain variable fragment), a transmembrane domain, and an intracellular sequence
Summary/Conclusion
T cells experience large swings in energy demand during the transition from naïve cells, to highly proliferative effectors, and back to quiescent memory cells. During this course, metabolic processes are tightly regulated to meet energetic outputs, and perturbations in this balance can affect not only rates of T cell proliferation, but also influence differentiation and effector function. Given the degree of these dramatic changes, and the fact that different metabolic pathways are active in
Conflicts of interest
The authors declare no conflicts of interest.
Funding
This work was supported by grants to CAB from the National Institute of Health – NHBLI. (1K08-HL123631), Children's Hospital of Pittsburgh Research Advisory Committee, the University of Pittsburgh Physicians Academic Foundation, the Hyundai Motor Company (Hope on Wheels Scholar grant), and the American Society of Hematology (Scholar award).
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