Transcriptional programing of T cell metabolism by STAT family transcription factors

T cells adapt their metabolism to meet the energetic and biosynthetic demands imposed by changes in location, behavior, and/or differentiation state. Many of these adaptations are controlled by cytokines. Traditionally, research on the metabolic properties of cytokines has focused on downstream signaling via the PI3K‐AKT, mTOR, or ERK‐MAPK pathways but recent studies indicate that JAK‐STAT is also crucial. This review synthesizes current thinking on how JAK‐STAT signaling influences T cell metabolism, focusing on adaptations necessary for the naïve, effector, regulatory, memory, and resident‐memory states. The overarching theme is that JAK‐STAT has both direct and indirect effects. Direct regulation involves STATs localizing to and instructing expression of metabolism‐related genes. Indirect regulation involves STATs instructing genes encoding upstream or regulatory factors, including cytokine receptors and other transcription factors, as well as non‐canonical JAK‐STAT activities. Cytokines impact a vast range of metabolic processes. Here, we focus on those that are most prominent in T cells; lipid, amino acid, and nucleotide synthesis for anabolic metabolism, glycolysis, glutaminolysis, oxidative phosphorylation, and fatty acid oxidation for catabolic metabolism. Ultimately, we advocate the idea that JAK‐STAT is a key node in the complex network of signaling inputs and outputs which ensure that T cell metabolism meets lifestyle demands.


Introduction
Cytokines profoundly impact T cell metabolism. Until recently, that capacity has been attributed mainly to downstream signaling via the PI3K-AKT, mTOR, or ERK-MAPK pathways but it is now clear that JAK-STAT is also critical. Most research on JAK-STAT in T cell metabolism has focused on canonical signaling whereby cytokine-driven receptor oligomerization activates JAK kinases which, in turn, phosphorylate STATs and thereby prompt dimerization, nuclear translocation and, ultimately, DNA binding via consensus STAT motifs ( Fig. 1) [1]. However, there is now growing appreciation for non-canonical signaling, particularly the role of mitochondrial STATs (Fig. 1) [2]. Here, we examine how both Receptor oligomerization prompts trans-activation of JAKs which, in turn, phosphorylate the cytoplasmic tails of receptors to create requisite docking sites for STATs. JAKs then mediate tyrosinephosphorylation of STATs (p-Tyr), leading to dimerization, nuclear translocation, DNA binding, and, ultimately, gene transcription. Canonical JAK-STAT signaling influences T cell metabolism in both direct and indirect ways. Direct regulation involves STATs localizing to and controlling expression of genes encoding enzymes, transporters, and accessory proteins involved with metabolism pathways. Indirect regulation involves STATs localizing to and controlling expression of transcription factors, cytokine receptors, and other genes that, in turn, control expression and/or function of metabolism pathway components. A role for non-canonical JAK-STAT signaling has also become apparent, particularly serine-phosphorylated STATs (p-Ser) within the mitochondrion.
Prior to antigen encounter, T cells persist in a 'naive' quiescent state (T nv ). They are motile, actively seeking antigen, but biosynthesis and energy consumption rates are low, the latter fueled primarily by oxidative phosphorylation (OxPhos). Upon antigen encounter, they transition from this naive state, focused on catabolic energy production, to an 'effector' state (T eff ) characterized by heightened catabolism and anabolism. Nutrient uptake is massively induced and energy production is both augment and diversified to meet demands imposed by growth, proliferation, and metabolically expensive effector functions, like cytokine production and cytolysis. There is also a corresponding rise in de novo synthesis of amino, fatty, and nucleic acids, again, to satiate demands imposed by growth, proliferation, and effector functions. Prompted by increased glucose and glutamine import, glycolysis and glutaminolysis are each turned up to feed the citric acid cycle, methionine cycle, and folate cycle, as well as to supply biochemical intermediates for amino acid and nucleic acid synthesis. At the same time, OxPhos is optimized and fatty acid oxidation (FAO) is diminished in favor of fatty acid synthesis (FAS). Both increased OxPhos and the switch from FAO to FAS are closely tied to increased mitochondrial mass and frequency, as well as to changes in mitochondrial structure, particularly a shift toward fissed mitochondria with loose cristae [9]. OxPhos is far more efficient than glycolysis at generating ATP but glycolysis is 100 times faster. Thus, both contribute substantively to energy production in effector T cells (T eff ), particularly during early stages of activation [5]. Glutaminolysis also contributes to energy production and feeds metabolic pathways that are important for T eff , including amine and amino acid synthesis [10,11], as do other ancillary metabolic processes, like hexosamine synthesis and the pentose phosphate pathway [12].
As antigen wanes, T eff either undergo apoptosis or differentiate toward a long-lived "memory" state (T mem ) with stem-like properties. With this transition comes a return to quiescence whereby anabolic metabolism is reduced, glycolysis is turned off and energy is again supplied by OxPhos. FAO also becomes a key energy source. Alternatively, if antigen does not wane, T eff may instead proceed to a dysfunctional 'exhausted' state and there is evidence that sustained glycolysis contributes to that outcome, which is deleterious in the context of chronic infection and cancers [13]. Ultimately, the metabolic profile of T mem becomes similar to that of T nv , although T mem also have distinct features which allow them to both persist and quickly return to an effector state. For instance, T mem are more reliant on FAO and are characterized by fused mitochondria which enable the acquisition of spare respiratory capacity to support long-term survival [14,15]. They are also able to trigger glycolysis more quickly than T nv , in part due to increased activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [16]. Another notable feature of T mem is that they continue to engage in FAS despite ramping up FAO, meaning that they simultaneously produce and break down lipids. The purpose of this 'futile' cycle remains unclear [3]. Importantly, the above chain of events-from naive to effector to memory-is evident for both human and mouse T cells and characteristic of both the CD4 + 'helper' and CD8 + "cytotoxic" lineages. They exhibit similar metabolic changes subject to many of the same upstream signals, including cytokines, the PI3K-AKT and mTOR pathways, and transcription factors like MYC, HIF1α, and IRF4 [8]. However, there are also notable differences, some of which are discussed below.
Beyond differentiation state, T cell metabolism is heavily influenced by local environment. Accordingly, resident memory T cells (T rem ), which inhabit mucosal interfaces like skin and gut, are metabolically distinct from lymphoid resident T eff and T mem [5,17,18]. Local environment also influences the metabolism of T reg , a subset of CD4 + T cells that are specialized to suppress pathogenic inflammation [19,20], as well innate lymphoid cells (ILC) [21] and macrophages [17,22,23], all of which may inhabit the same anatomical niches [5,17]. Regarding the latter, the distinction between pro-inflammatory M1 macrophages, which rely on glycolysis, and anti-inflammatory M2 macrophages, which rely on OxPhos and FAO, has been extensively studied, with recent studies arguing against strict metabolic dichotomy [17,22,23]. It is also important to note that T reg deviate from the canonical T cell metabolism program in that they exhibit both T eff and T memlike properties. Similar to T eff , activated T reg engage in glycolysis and glutaminolysis while, similar to T mem , resting T reg have low anabolic rates and generate energy primarily by OxPhos and FAO [20]. The basic principles of T cell metabolism also apply to other lymphoid lineages. The overarching theme is that when lymphocytes are in quiescent states, as with T nv and T mem , nutrient uptake and biosynthesis rates are low and energy is supplied mainly by OxPhos and FAO. When they are in activated states, as with T eff , nutrient uptake, and biosynthesis rates are high and increased energy demand is met by a combination of glycolysis and OxPhos. Thus, resting B cells have low biosynthesis rates and mainly use OxPhos for energy production, germinal center, and plasma cells have high biosynthesis rates and use both OxPhos and glycolysis, while memory B cells return to low biosynthesis rates, shut down glycolysis and, again, rely on OxPhos and FAO [24].

STATs directly instruct T cell metabolism
STATs are classical transcription factors (TF) in that they bind DNA regulatory elements, like promoters and enhancers, and thereby modulate gene transcription. Specifically, they are "signaldependent" TF as they must first be phosphorylated by upstream signals (i.e., JAKs) [25]. It is now understood that STATs directly engage and control expression of numerous genes encoding enzymes, transporters, and co-factors involved with metabolic processes (Fig. 1). In fact, paired surveys of genome-wide STAT occupancy (by ChIP-seq) and STAT-dependent gene expression (by RNA-seq) have established that hundreds of metabolismrelated genes may be subject to direct transcriptional control in CD4 + T cells [26][27][28][29][30]. This is in contrast to the PI3K-AKT and mTOR pathways, which mainly operate by posttranscriptional mechanisms [31, 32], but in line with the idea that transcriptional regulation of metabolism is also crucial [8]. Thus a stepwise model emerges whereby STATs instruct transcription of certain metabolism-related genes, then PI3K-AKT and mTOR act on the resulting gene products (i.e., mRNAs, proteins) while also performing other critical metabolic functions.
The role of STAT3 in glycolysis appears context-dependent. On the one hand, it can suppress glycolysis and several mechanisms have been proposed for this effect downstream of IL-10 in M1-type macrophages. These include direct instruction of the DDIT4 locus, which encodes an inhibitor of the mTOR pathway, direct suppression of glycolysis-associated genes, and blocking transit of the glucose transporter, GLUT1, to the plasma membrane [38]. In addition, by promoting M2 macrophage differentiation at the expense of M1, STAT3 pushes cells toward a fate that is inherently more dependent on FAO and OxPhos than glycolysis [42]. STAT3 is also known to inhibit glycolysis in tumorinfiltrating CD8 + T eff cells, suggesting a mechanism whereby it limits protective anti-tumor responses [35]. However, there are also numerous settings where STAT3 appears promote glycolysis, most notably in cancer cells, where it directly engages loci encoding key pathway elements, including LDHA and HK2 [43]. STAT3-activating cytokines have also long been associated with oxidative metabolism in myeloid cells [33] and similar activities in T cells and other lymphoid lineages have now been reported (Fig. 2). For instance, STAT3 is required for cytokine-driven FAO in CD8 + T eff cells, where it binds and instructs transcription of the loci encoding rate-limiting enzymes, CPT1A and CPT1B [34,35]. An analogous system is evident in breast cancer stem cells, where it promotes FAO downstream of leptin via induction of CPT1B [36]. STAT3 also promotes OxPhos in multiple immune lineages, In T cells, canonical STAT3 signaling is typically invoked by JAK1 and JAK2 downstream of GP130 or IL-10 family cytokines to support catabolic metabolism, specifically Fatty Acid Oxidation (FAO), Glutaminolysis, and Oxidative Phosphorylation (OxPhos). Canonical STAT5 signaling is typically invoked by JAK3 downstream of cγ family cytokines to support both catabolic and anabolic metabolism. STAT5driven catabolism pathways include FAO, Glutaminolysis, OxPhos, and glycolysis. STAT5-driven anabolic pathways include Fatty Acid, Amino Acid, and Nucleotide Synthesis.

STAT5 as a master regulator
STAT5 is a major driver of both anabolic and catabolic metabolism in T cells (Fig. 2). Upstream cytokines, particularly those employing the common gamma (cγ) receptor, have long been studied in the context of T cell metabolism and a model has emerged whereby different cγ cytokines operate across differentiation states, depending on which co-receptors are expressed [44]. Quiescent T nv bear high levels of IL-7 receptor (IL-7R) and, thus, IL-7 is a major influence on their metabolism. Importantly, downstream STAT5 signaling instructs transcription of GLUT1, fueling basal glycolysis and OxPhos [45]. Activated T eff lose IL-7R but gain IL-2R, IL-4R, IL-9R, IL-15R, and/or IL-21R. Most of these are broadly expressed across effector subsets but some are circum-scribed, like IL-4R, which is most abundant in Th2-type T eff . IL-2 is the cγ family member that has been most extensively studied in the context of T eff metabolism and as discussed below, a key role for downstream STAT5 signaling is now evident. Quiescent T mem re-express IL-7R but also retain other co-receptors and, thus, are generally better able to respond to cγ cytokines than naive counterparts. One important function for IL-7 in T mem that is likely driven by STAT5 is the induction of the glycerol channel, aquaporin 9 (AQP9), which helps fuel FAO [14]. Intriguingly, this does not appear to occur in T nv , which also express IL-7R but are less reliant on FAO [14].
T rem are diverse in terms of cγ co-receptors as expression levels vary based on anatomical location, activation status and numerous other upstream factors [44]. Nevertheless, IL-15 has emerged as a 'universal' driver of T rem metabolism and, likely, STAT5 is a key downstream mediator [5,17]. For instance, STAT5 likely mediates IL-15-driven induction of CPT1A, TFAM, and OPA1 in T rem , thereby promoting FAO, mitochondrial respiratory capacity, and mitochondrial fusion [3,8]. Like T rem , T reg also express multiple cγ co-receptors [44]. However, most studies on T reg metabolism have focused on IL-2 because they are conspicuously marked by the high-affinity IL-2R and, thus, exquisitely sensitive to its effects [19]. IL-2 induces transcription of numerous metabolismrelated genes in T reg but, crucially, these mostly differ from those induced in T eff . Notably, IL-2 does not readily induce transcription of genes involved with glycolysis in T reg but does in T eff , crucially, via STAT5-driven induction of key pathway elements[30].

The IL-2-STAT5 axis
IL-2 is the emblematic example of a STAT-activating cytokine with overt metabolic activities. Its role in T cell metabolism has been studied for over 30 years but, as noted, that research has traditionally focused on downstream PI3-AKT and mTOR signaling. However, it is now apparent that STAT5 is also critical and, in fact, required for parallel mTOR activity [30]. For details on how PI3-AKT and mTOR influence cellular metabolism, we refer to comprehensive reviews [31,46]. Readers should also be aware that there is ongoing debate about whether and how IL-2 propagates PI3K-AKT signaling in T cells, with recent evidence indicating that it does not [47]. Another important consideration is that, since T cells produce IL-2 quickly after antigenic stimulation, it can be difficult to distinguish metabolic events driven by IL-2, from those driven by the T cell receptor (TCR) and/or co-stimulation (CoStim). That difficulty is compounded by the fact that IL-2 and TCR/CoStim each propagate mTOR and PI3K-AKT signaling. Thus, current models posit that TCR and CoStim act first during the earliest stages of T eff differentiation and are then subsequently joined by IL-2, which first enforces TCR/CoStim-driven processes and then maintains them as TCR/CoStim wanes [5,6].
At least four major themes have emerged from research on how IL-2 shapes T cell metabolism. Together, they point to a central role in transition from the quiescent naive state to the highly energetic effector state. First, IL-2 rapidly and potently induces uptake of combustible carbohydrates, particularly glucose and glutamine, and nutrients like amino acids [47]. In almost all cases, there is evidence that downstream STAT5 signaling instructs expression of relevant transporters, such as GLUT1, SLC1A5, the principal transporter for glutamine, and SLC7A5, the principal transporter for essential amino acids (Fig. 1) [30, 45,48,49]. Second, IL-2 rapidly and potently induces glycolysis, glutaminolysis, and OxPhos, enabling cells to burn their newly obtained fuels [47,50,51]. Several enzymes in these pathways are directly regulated by STAT5 [30]. Third, IL-2 promotes macromolecule synthesis, thereby providing necessary building blocks for cellular growth and proliferation. Again, there is evidence that STAT5 is intimately involved, with recent studies showing that it instructs transcription of key genes involved with fatty, amino, and nucleic acid synthesis [30]. Fourth, IL-2 promotes epigenetic remodeling of metabolism-related gene loci, in part, by inducing necessary substrates for epigenetic modifications. For instance, the IL-2-STAT5 axis promotes DNA and histone methylation by promoting accumulation of alpha-ketoglutarate [52][53][54]. Given these and other striking effects, it is no wonder that IL-2 continues to be viewed as a key motivator of T cell metabolism.
As mentioned, the role STAT5 in IL-2-driven T eff metabolism has now been systematically addressed [30]. By integrating genome-, transcriptome-and metabolome-wide analyses, it was established that STAT5 localizes to enhancers and promoters for >90 genes encoding essential, often rate-limiting enzymes, transporters, and co-factors, where it instructs transcriptional activity through p300 recruitment and epigenetic landscaping. These effects were most evident within the glycolysis, OxPhos, and amino acid synthesis pathways. It was also reported that STAT5 is required for and, therefore, upstream of mTOR signaling, and for both expression and function of MYC, a TF with wide-ranging metabolic activities. Together, these findings emphasize the role of STAT5 in powering T eff metabolism downstream of IL-2.

Beyond STAT3 and STAT5
Despite their prominence, cγ cytokines are not the only relevant STAT5 stimuli for T cells [1]. In fact, STAT5 is a bottleneck for numerous upstream receptors, many of which are targeted by popular small molecule JAK and Receptor Tyrosine Kinase (RTK) inhibitors [55]. Also, beyond STAT3 and STAT5, other STAT family members can impact T cell metabolism and, in most cases, there is evidence for direct engagement of relevant gene loci. For instance, STAT1 promotes FAO and OXPHOS downstream of type I IFN in plasmacytoid DCs, glycolysis downstream of type II IFN in macrophages and is known to instruct transcription of cholesterol 25-hydroxylase, a key enzyme in the generation of oxysterols, and IDO1, an enzyme involved with tryptophan catabolism [56]. STAT2 is activated downstream of type I interferons and is likely required for most (if not all) of their metabolic effects. Notably, STAT2 dimerizes with STAT1 prior to JAK-mediated phosphorylation, which then reorients dimers to enable canonical signaling. It is not thought to dimerize with any other STAT or to participate in the signaling of any other cytokine [1]. STAT4 activation is also highly restricted, evident mainly downstream of a subset of IL-6/IL-12 family cytokines. Two of these, IL-12 and IL-23, are major players in T cell biology and IL-12 is known to induce glycolysis and OxPhos in both NK cells and T eff [11,[57][58][59]. At least one set of relevant target genes has been codified; STAT4 binds and instructs genes encoding SREBP proteins, a set of transcription factors involved with cholesterol synthesis and other vital metabolic processes, including OxPhos (Fig. 1) [60]. STAT6 is mobilized primarily downstream of IL-4 and IL-13, although there is growing appreciation for other stimuli, like the cGAS-STING pathway [1]. It promotes glycolysis in both B cells and T cells, in part, by instructing transcription of GLUT1 [24,61], but this is likely only the tip of the iceberg as IL-4 is known to modulate expression of many genes involved with cellular metabolism and STAT6 is responsible for much of the transcriptional output downstream of IL-4 [29]. The same can be said of IL-13 and, specifically, its ability to promote OxPhos and FAO in M2 macrophages [62].
Along with metabolism, STATs also instruct T cell differentiation. This has been studied most extensively in CD4 + helper T cells, where every STAT (save STAT2) is linked to at least one effector subset as defined by stereotypical patterns of transcription factor and cytokine expression [63]. For instance, STAT1 and STAT4 are linked to Th1-type effectors expressing the transcription factor T-BET and the cytokine IFN-γ, STAT3 is linked to Th17type effectors expressing RORγt and IL-17, and STAT6 is linked to Th2-type effectors expressing GATA-3, IL-4, IL-5, and IL-13. STAT5 is linked to both Th1-and Th2-type effectors and is a potent inhibitor of Th17-type effectors. Induction of metabolism pathways, most notably glycolysis, glutaminolysis, OxPhos, and FAO, also varies across subsets [58,[64][65][66][67]. These disparities are often tied to the cytokines used for subset polarization (i.e., lineage-specifying cytokines) so it is likely that STATs are involved. Th17-type effectors offer an intriguing example. STAT3-activating cytokines, particularly IL-6 and IL-23, promote Th17 differentiation at the expense of Th1 and Treg differentiation, so it stands to reason that they also underlie selective induction of glutaminolysis, with then further reinforces the Th17 differentiation program [10,11,68].

STATs in secondhand regulation
A more indirect way that STATs influence T cell metabolism is by controlling expression of other transcription factors with overt metabolic activities (Fig. 1). MYC, HIF1α, and IRF4 are prime examples. MYC expression is low/absent in T nv and T mem but high in T eff , where it controls a wide range of metabolic processes, including nutrient import, glycolysis, glutaminolysis, OxPhos, and FAO [69,70]. Importantly, STAT3 and STAT5 each bind and induce transcription of the MYC locus [40,71,72]. In T cells, MYC expression is closely tied to IL-2 and prevailing dogma is that MYC is the principal downstream mediator of its metabolic activities. However, as discussed, recent evidence indicates that STAT5 signaling impacts T cell metabolism beyond its ability to induce MYC and, in fact, is required for MYC-driven transcription of metabolism-related genes [30]. Like MYC, HIF1α, and IRF4 each promote glycolysis and influence other metabolism pathways [8]. Also like MYC, multiple STATs are known to bind and instruct their respective gene loci, with STAT3 considered a key regulator [73][74][75]. STAT3 is also considered a "pioneer" factor for IRF4 activity, meaning that it precedes and enables IRF4 binding to sites throughout the genome [76]. Thus, STATs not only induce expression of metabolism-related transcription factors but also enable them to execute metabolic functions.
STATs also control expression and/or function of transcription factors whose role in cellular metabolism is less overt. One striking example is BCL6 (Fig. 1). It has long been known that BCL6 expression is controlled by STATs-STAT3 and STAT4 directly induce it while STAT5 directly inhibits it-and a key role in T cell metabolism is now apparent [77]. BCL6 restrains transcription of numerous genes involved with glycolysis, including GLUT1, PKM, and HK2, in part, by antagonizing positive regulation via MYC and T-BET, another transcription factor whose expression is STAT-driven [78]. PRDM1 is also relevant in this context, as it is induced by STAT5, antagonizes BCL6 and, thus, is a positive regulator of glycolysis [51]. A major theme emerging from this work is that changes in T cell metabolism require coordination between JAK-STAT signaling and downstream transcription factors, some of which are also critical for T eff differentiation (e.g., BCL6, T-BET) and, thus, link particular metabolism pathways to particular effector subsets.
Another way that STATs modulate T cell metabolism is by enabling or disabling cytokine receptor signaling and thereby enabling or disabling downstream STAT-dependent andindependent events (Fig. 1). Perhaps the most famous example is the ability of STAT5 to induce transcription of IL-2Rα and thereby license IL-2 signaling [79]. Others include the ability of STAT1 to induce IL-12R, the ability of STAT3 to induce IL-6R, and the ability of STAT5 to induce IL-4R [79]. STATs also form positive feedback loops whereby they induce their own expression and enforce transcription of STAT target genes [80]. Thus, STATs control T cell metabolism by a variety of direct and indirect means, all in their guise as classical transcription factors.

Non-canonical JAK-STAT signaling
JAK-mediated p-Tyr of the transactivation domain is the canonical instigating event for JAK-STAT signaling. However, there are also other posttranslational modifications (PTMs) that can influence STAT function, including glycosylation, methylation, acetylation, and serine phosphorylation [80]. PMTs temper or enhance STAT activity and, thus, modulate their ability to influence metabolism pathways. For instance, serine phosphorylation promotes STAT3 activity and, in turn, STAT3-driven glycolysis in LPS-stimulated macrophages [81]. It is also notable that metabolism pathways control availability of biochemicals necessary to enact PMTs. For instance, glycolysis and glutaminolysis limit availability of UDP-GlcNAc, which is needed for glycosylation, and the citric acid cycle generates acetyl-CoA and alpha-ketoglutarate, which are needed for acetylation and methylation, respectively [52,82].
Another non-canonical aspect of the JAK-STAT pathway that likely impacts T cell metabolism involves the ability of STAT3, and perhaps STAT5 [83], to localize to the mitochondrion (Fig. 1). Seminal work by the Levy and Larner laboratories discovered that serine (but not tyrosine) phosphorylated STAT3 enters the mitochondria to promote OxPhos [84,85]. Later studies revealed that mitochondrial STAT3 (mitoSTAT3) also influences pyruvate metabolism and redox balance [86,87]. These properties have mainly been studied in cancer cell lines, where it is known to physically interact with multiple Electron Transport Chain components [88]. However, the stoichiometry of mitoSTAT3 versus ETC components makes it unlikely that this is a primary mechanismof-action [89] and there is evidence that mitoSTAT3 also interacts with pyruvate dehydrogenase complex E1 and cyclophilin D, and that it drives transcription of the mitochondrial genome [86,87,90]. As alluded, the role of mitoSTAT3 in immune cells has not been extensively studied but early work suggests that it does impact T cell motility and migration [91].

Perspectives
Much of what has been learned about T cell metabolism is from in vitro studies. This reductionist approach has obvious advantages, like the ability to precisely control culture conditions, to strictly define start and end points, and to scale up for inputintensive biochemical assays. Considering that adoptive T cell therapies, particularly TIL and CAR therapies, use JAK-STATactivating cytokines for in vitro expansion and/or differentiation, it is clear that this work has practical value. For instance, understanding which metabolic pathways are subject to STATs may allow for coupling pathway targeting drugs (e.g., JAK inhibitors, recombinant cytokines) with regiments intended to supplement or deplete certain metabolites. However, it is also certain that in vitro cultures do not fully replicate the complexity of intact biological systems. This latter point is particularly relevant for T rem as numerous studies have shown that the local environment dictates their metabolic requirements [5,17,92]. Thus, while we have focused on cellular metabolism, we must also recognize the reciprocal relationship between cellular, tissue, and organismal metabolism; each influences one another. It is also important to note that STATs are activated by many factors with tissue-and organism-level effects, including leptin, insulin and growth hormone. Therefore, it is not surprising that STATs have emerged as major players in higher-order metabolism pathways. Notable examples include the role of STAT3 and STAT6 in beige fat neogenesis [93,94], STAT5 in controlling triglyceride levels [95], and STAT3 in controlling systemic glucose levels [96]. This connection will only strengthen moving forward as the field of metabolism research moves toward in vivo biology, with the JAK-STAT pathway now in the foreground, together with PI3K-AKT, mTOR, or ERK-MAPK.