能量免疫：洞察代谢和淋巴细胞功能

所翻译综述发表于Science的时间：2013-10-11 影响因子：31.027

译者：谢金攀 翻译完成时间：2013-12-20

REVIEW

综述

Fueling Immunity: Insights into Metabolism and Lymphocyte Function

能量免疫：洞察代谢和淋巴细胞功能

Erika L. Pearce, Maya C. Poffenberger, Chih-Hao Chang, Russell G. Jones

Lymphocytes face major metabolic challenges upon activation. They must meet the bioenergetic and biosynthetic demands of increased cell proliferation and also adapt to changing environmental conditions, in which nutrients and oxygen may be limiting. An emerging theme in immunology is that metabolic reprogramming and lymphocyte activation are intricately linked. However, why T cells adopt specific metabolic programs and the impact that these programs have on T cell function and, ultimately, immunological outcome remain unclear. Research on tumor cell metabolism has provided valuable insight into metabolic pathways important for cell proliferation and the influence of metabolites themselves on signal transduction and epigenetic programming. In this Review, we highlight emerging concepts regarding metabolic reprogramming in proliferating cells and discuss their potential impact on T cell fate and function.

淋巴细胞在活化时面临主要的代谢挑战。它们必须满足增长的细胞增殖对生物能量和生物合成的要求，并且适应营养和氧气可能受限的变化中的环境条件。免疫学一个新兴的主题是，代谢重编程和淋巴细胞活化有复杂的联系。不管怎样，为什么T细胞采用特定的代谢程序，这些程序对T细胞功能的影响，以及最终的免疫结果都仍不明确。关于肿瘤细胞代谢的研究已经提供了有价值的洞见，涉及细胞增殖的重要代谢途径和代谢本身对信号转导和表观遗传学编程的影响。在这篇综述，我们突出关于增殖细胞中代谢重编程新兴的概念并探讨它们对T细胞命运和功能的潜在影响。

The immune system is comprised of a series of specialized cells conditioned to respond rapidly to“danger”signals such as foreign pathogens or inflammatory stimuli. T lymphocytes,or T cells, are sentinels of the adaptive immune system that respond to antigen-specific signals by blasting, proliferating, and differentiating into effector subsets tailored to identify and eliminate threats to the host. Integrated into this program of activation is the regulation of cellular metabolism.Upon activation, T cells dramatically alter their metabolic activity to meet the increased metabolic demands of cell growth, proliferation, and effector function. Metabolism fundamentally underpins T cell function; thus, there is great interest in understanding how metabolic pathways influence immune responses and ultimately affect disease progression.It should be noted that“metabolism”refers to a complex network of biochemicalreactions involved in energy production and macromolecular biosynthesis, and comprehensive coverage of such a broad topic is difficult. Several recent reviews have highlighted the molecular mechanisms that govern metabolic reprogramming in the immune system (1–3). This Review will focus on emerging areas in intermediary metabolism in lymphocytes and will discuss their potential impact on T cell fate, plasticity, and effector

function.

免疫系统由一系列有条件快速应答例如外来病原体或者炎症刺激物等“危险”信号的专门细胞组成。T淋巴细胞或者T细胞，是应答抗原特异性信号的适应性免疫的哨兵，因为它们爆发、增殖并分化为识别和清除对宿主的威胁的定制的效应细胞亚群。细胞代谢的调节被整合到活化的程序。在活化时，T细胞戏剧性地转变它们的代谢活动来满足细胞生长、分化及效应细胞功能所增加的代谢需求。代谢从根本上支撑T细胞功能；因此，搞明白代谢途径怎样影响免疫应答并最终影响疾病进展会很让人感兴趣。应当指出的是，“代谢”指一个涉及能量产生和大分子生物合成的复杂生化反应网络，而全面覆盖这样一个宽的话题是困难的。最近的几篇综述已经强调了免疫系统中控制代谢重编程的分子机制(1–3)。本篇综述将关注淋巴细胞中间代谢的新兴领域，并将探讨它们对T细胞命运和效应细胞功能的潜在影响。 Differential Regulation of T Cell Metabolism

T细胞代谢的不同调节

Lymphocyte Metabolism Is Dynamically Regulated

淋巴细胞代谢被动态地调节

Maintenance of cellular bioenergetics is an essential function of all living cells, and lymphocytes are no exception. In T lymphocytes, glucose is a critical substrate for adenosine triphosphate(ATP) production (4). During glycolysis, glucose is broken down into two molecules of pyruvate.This process, which does not require oxygen,yields two reduced nicotinamide adenine dinucleotide (NADH) molecules and two net ATP molecules per molecule of glucose. Pyruvate has two alternate fates. Most terminally differentiated, nonproliferating cells can fully oxidize pyruvate in the tricarboxylic acid (TCA) cycle. This process generates NADH and reduced flavin adenine dinucleotide (FADH2), which the cell can use to fuel OXPHOS, an oxygen-dependent process that produces up to 36 molecules of ATP per glucose molecule. Alternatively, pyruvate can be transformed (or fermented) into lactate, regenerating NAD+ for subsequent use in glycolysis (5). From a bioenergetic perspective, engaging OXPHOS maximizes the amount of ATP that can be derived from glucose.

细胞的能量维持是所有活细胞的一项必要的功能，并且淋巴细胞也不例外。在T淋巴细胞，葡萄糖是三磷酸腺苷（ATP）产物的关键底物(4)。在糖酵解中，葡萄糖被分解为两分子丙酮酸。这个不需要氧的过程，每分子葡萄糖产生两分子还原型烟酰胺腺嘌呤二核苷酸(NADH)和两个净ATP分子。丙酮酸有两种可选择的命运。大部分终末分化的非增殖细胞能在三羧酸循环中充分氧化丙酮酸。该过程产生NADH和还原型黄素腺嘌呤二核苷酸(FADH2)，细胞能用它给氧化磷酸化加料，该氧依赖的过程中每分子葡萄糖产生大于36个ATP分子。另外，丙酮酸能被转化（或者发酵）为乳酸，再生NAD＋给随后的糖酵解利用(5)。从生物能学这一观点来看，参与的氧化磷酸化最大化能来自葡萄糖的ATP数量。

Bioenergetic profiling of T cells has revealed that T cell metabolism changes dynamically with activation state (Fig. 1). Upon antigen encounter,T cells become activated, undergo extensive proliferation, and differentiate into effector T cells(TEFF); upon pathogen clearance, most (TM). Consistent with the metabolism of othernonproliferating cells,resting na?ve T cells (T cells that have not yet encountered antigen) maintain low rates of glycolysis and predominantly oxidize glucose-derived pyruvate via OXPHOS or engage fatty acid oxidation (FAO) to make ATP. Upon activation, T cells switch to a program of anabolic growth and biomass accumulation to generate daughter cells, which by definition dictates increased demand for ATP and metabolic resources. In this state,T cells are considered to be metabolically activated (Fig. 1).

T cell receptor (TCR) signaling directs the metabolic reprogramming of na?ve T cells. TCR ligation promotes the coordinated up-regulation of glucose and amino acid transporters (6–8), facilitating nutrient uptake and T cell blastogenesis. TCR-mediated up-regulation of the transcription factors c-Myc (9) and estrogenrelated receptor α(ERRα)(10) enhances the expression of genes involved in intermediary metabolism. In addition, catabolic pathways of ATP generation such as fatty acidβ-oxidation are actively suppressed (9). The predominant metabolic phenotype of activated T cells is a shift to aerobic glycolysis [reviewed in (11)]. Both CD4+ and CD8+TEFF cells engage aerobic glycolysis,which is marked by the conversion of glucose derived pyruvate to lactate despite the availability of oxygen for complete glucose oxidation. This process, also known as the Warburg effect from earlier work in cancer biology, is a common trait of actively proliferating cells (5). It is important to note that OXPHOS is still engaged in TEFF cells (9); however, the production of lactate from pyruvate by aerobic glycolysis is the dominant pathway of glucose metabolism in TEFF cells. Regulating energy metabolism may provide a way for T cells to reversibly switch between quiescent and highly proliferative states (12).

T细胞生物能学的侧面已显露出T细胞的代谢随着活化状态而动态地改变（图1）。当遇到抗原时，T细胞变得活化，经历广泛的增殖并分化为效应T细胞(TEFF)；当抗原清除时，大部分的细胞死掉，留下少量长命的抗原特异性记忆T细胞(TM)。与其它非增殖的细胞的代谢一致，静止的原始T细胞（那些还没遭遇抗原的T细胞）保持低比率的糖酵解并主要通过氧化磷酸化氧化来自葡萄糖的丙酮酸或者进行脂肪酸氧化(FAO)来产生ATP。一旦活化，T细胞转向一个合成代谢的生长和生物量的累积的程序来产生子代细胞，这基于增加ATP和代谢原料的要求的定义指令。在这状态，T细胞被认为是代谢上的活化（图1）。T细胞受体(TCR)发信号指导原始T细胞的代谢重编程。TCR被结合促进葡萄糖和氨基酸转运载体的同步上调(6–8)，推进营养摄取和T细胞母细胞化。TCR介导的转录因子c-Myc (9)上调和雌激素相关性受体α(ERRα)(10)增强参与中间代谢的基因的表达。此外，ATP合成的分解代谢途径被积极抑制，如β脂肪酸氧化(9)。活化的T细胞主要的代谢表型是转向有氧糖酵解[综述于(11)]。CD4+和CD8+的TEFF细胞都进行有氧糖酵解，其标志是尽管有足够的氧进行葡萄糖的完全氧化，仍将葡萄糖来源的丙酮酸转化为乳酸。该过程也被称为Warburg效应，源于肿瘤生物学的早期工作，这也是活化增殖细胞的共同特点(5)。应重要注意氧化磷酸化仍存在于TEFF细胞(9)；不管怎样，通过有氧糖酵解从丙酮酸产生乳酸是TEFF细胞的主要葡萄糖代谢途径。调节能量代谢可能提供T细胞在静止和高度增殖的状态之间可逆性转变的一种方式(12)。

As a quiescent T cell population, TM cells adopt a metabolic profile similar to that of na?ve T cells—a catabolic metabolism characterized by increased reliance on OXPHOS and lower rates of nutrient uptake and biosynthesis relative to TEFF cells (Fig. 1). However, TM cells also display a characteristic increase in mitochondrial mass, which translates into greater mitochondrial spare respiratory capacity (SRC) relative to na?ve or TEFF populations (13). SRC can be viewed as the maximal respiratory capacity available to a cell, much like the maximum speed that can be achieved by a car engine. Under increased workload, stress, or nutrient limitation, cells engage this reserve capacity to generate more energy and promote cell viability (14,15). We have recently shown that increased mitochondrial mass and SRC of TM cells allows for rapid mitochondrial ATP production upon TCR engagement, conferring a bioenergetic advantage to TM cells upon secondary exposure to antigen (16). From this vantage, TM cells may be viewed as being metabolically primed, with mitochondrial

metabolism fueling the rapid recall response to reinfection.The memory T cell–promoting cytokine interleukin (IL)–15 plays a key role in this catabolic switch by promoting mitochondrial biogenesis (13).

作为静止的T细胞群，TM细胞采用类似于原始T细胞的代谢模式（metabolic profile）——具有相对于TEFF细胞更依赖氧化磷酸化和更低比率的营养摄取及生物合成的特点的分解代谢（图1）。然而，TM细胞也显示线粒体物质的特征性增加，这转化为相对于原始的或TEFF 群更强的线粒体呼吸储备能力(SRC) (13)。SRC能被视为对一个细胞有用的最大呼吸能力，很像一个汽车发动机能获得的最大速度。在增加的工作负担、压力或者营养限制下，细胞动用该储备能力来产生更多的能量并促进细胞的生存能力(14,15)。我们最近已经表明，在TCR 参与时TM细胞增加的线粒体物质和SRC允许快速的线粒体ATP产生，赋予在二次暴露于抗原时TM细胞的生物能学的优势(16)。从这优势出发，TM细胞可能被视为有代谢准备，在线粒体代谢下为再次感染的快速应答供能。促进记忆T细胞的细胞因子白介素(IL)—15通过促进线粒体生物合成在这个分解代谢的转换中起关键作用(13)。

The mechanisms governing the transition of T cells from effector to memory states are still poorly understood, but recent work hints that changes in metabolism may influence this process.We previously demonstrated that mitochondrial FAO stimulated downstream of TNF (tumor necrosis factor) receptor–associated factor 6 (TRAF6) is required for memory CD8+T cell development(17). Oxidation of free fatty acids (FFAs) generates acetyl–coenzyme A (CoA), which can be metabolized further in the TCA cycle, as well as FADH2 and NADH, which can be used directly by the electron transport chain (ETC) to make ATP. FFAs are energy-dense molecules, and FAO may be a preferred fuel source for TM cells as they rely on OXPHOS-dependent metabolic program. Administration of metformin, a metabolic stressor that activates the energy sensor adenosine monophosphate–activated protein kinase (AMPK),enhances the generation of CD8+T cell memory(17). One consequence of AMPK activation is the suppression of mammalian target of rapamycin complex 1 (mTORC1) activity in response to energetic stress (18). Consistent with this, the drug rapamycin, which also inhibits mTORC1, enhances the generation of CD8+TM cells (17,19,20). These observations suggest that manipulating the metabolism of antigen-specific cells during contraction can influence the development of TM cells.Given these observations, TM formation may be influenced by a number of enzymes and transporters involved in fatty acid synthesis, desaturation, and oxidation, as well as the availability of FFAs to memory precursor cells. Some important players to consider in this regard include acetyl-CoA carboxylase (ACC2) (21), the mitochondrial lipid transporter CPT1A (13, 22), and metabolites such as acetyl-CoA and malonyl-CoA (23).AMPK activation and mTOR inhibition are also both potent activators of autophagy, a catabolic process induced during starvation that promotes the degradation and recycling of cellular components [reviewed in detail in (24)]. Proper induction of autophagy has been shown to be important for the maintenance of cellular bioenergetics and sustained T cell viability after activation (25,26).It will be interesting to determine whether autophagy,by coupling catabolic fuel supply to mitochondrial metabolism, is important for TM formation after infection.

控制T细胞从效应状态转变为记忆状态的机制仍知之甚少，但最近的工作提示代谢中的改变可能影响该过程。我们先前证明线粒体FAO刺激的TNF（肿瘤坏死因子）受体相关因子6 (TRAF6)的下游为CD8+T记忆性细胞发展所需(17)。游离脂肪酸(FFAs)氧化产生乙酰辅酶A (CoA)，CoA在TCA循环中能被进一步代谢，同FADH2和NADH一样能被电子传递链(ETC)

直接用来产生ATP。FFAs是高能分子，而FAO可能是TM细胞所偏好的一种能量来源，因为它们依赖基于氧化磷酸化的代谢程序。二甲双胍是一种激活单磷酸腺苷活化的蛋白激酶(AMPK)的代谢应激物，施用二甲双胍增强CD8+T细胞记忆的产生(17)。一系列的AMPK活化将抑制对能量压力应答的哺乳动物靶向的雷帕霉素复合物1 (mTORC1)的活动(18)。与此一致，雷帕霉素药物也抑制mTORC1，增强CD8+T细胞的产生(17,19,20)。这些观察提示对缩减期抗原特异性细胞的代谢的操纵能影响TM细胞的发展。基于所给这些观察推测，TM 的形成可能受涉及脂肪酸合成、去饱和和氧化的许多酶及转运体所影响，就同游离脂肪酸对记忆前体细胞的有效性一样。从这角度考虑，重要的参与者包括乙酰辅酶A羧化酶(ACC2) (21)，线粒体脂质转运体CPT1A (13, 22)，以及例如乙酰辅酶A和丙二酰辅酶A的代谢产物(23)。AMPK活化和mTOR抑制物也都是自吞噬强有力的激活剂，自吞噬是饥饿时诱发的分解代谢过程，促进降解和细胞组成部分的回收再利用[详尽综述于(24)]。适当诱发自吞噬已被表明对细胞生物能学的维持和TM细胞活化后持续的生存能力很重要(25,26)。决定是否自吞噬将是有趣的，伴随分解代谢线粒体的能量供应，这对感染后TM形成是重要的。 Mitochondrial OXPHOS and T Cell Activation

线粒体的氧化磷酸化和T细胞活化

Although much focus has been placed on the shift toward glycolysis that accompanies T cell activation, evidence suggests that mitochondrial OXPHOS is also important for T cell activation.Oligomycin, a specific inhibitor of mitochondrial ATP synthase, can block the expression of early activation markers after TCR ligation and blunts subsequent T cell proliferation (27), suggesting that the na?ve-to-effector transition requires either de novo production of ATP by mitochondria or specific signals generated during mitochondrial ATP production. Mitochondrial-derived reactive oxygen species (ROS) may function as such a“bioenergetic”second messenger. There has long been evidence that ROS can play critical roles in shaping T cell responses (28–30). However, recent work suggests that mitochondrial ROS produced during OXPHOS is essential for T cell activation.T cells deficient for ubiquinol-cytochrome c reductase (Uqcrfs1), a component of complex III of the ETC, display impaired TCR-dependent ROS production and defects in antigen-specific proliferation (31). Intracellular calcium (Ca2+) flux, an early event in TCR signal transduction, may provide the functional link between TCR ligation,mitochondrial OXPHOS, and cell proliferation. Uptake of Ca2+ by mitochondria stimulates Ca2+-dependent dehydrogenases of the TCA cycle, driving mitochondrial NADH production and ATP production by OXPHOS during early T cell activation (32). T cells lacking the apoptosis regulators Bax and Bak, which display defects in intracellular Ca2+ homeostasis, exhibit reduced Ca2+-dependent mitochondrial ROS production and T cell proliferation after TCR stimulation (33).Restoring Ca2+ signals in Bax/Bak-null T cells restores mitochondrial ROS production and T cell proliferation (33). Thus, although toxic in many biological settings, mitochondrial-dependent ROS may prime T cells and license full T cell activation.

尽管许多焦点聚焦于向伴随T细胞活化的糖酵解方的转移，但证据提示线粒体的氧化磷酸化对T细胞活化也重要。寡霉素是线粒体ATP合酶的一种特异抑制剂，它能阻碍TCR接合后早期活化标志的表达并使T细胞的进一步增殖变得迟钝(27)，这提示原始细胞到效应细胞的转变需要线粒体ATP的重新产生或者线粒体ATP产生过程中生成的特殊信号。来自线粒体的活性氧(ROS)可能作为这样一个“生物能学的”第二信使起作用。长期以来有证明表明ROS能在形成T细胞应答中起关键作用(28–30)。不管怎样，最近的工作提示氧化磷酸化过程中产生的线粒体活性氧对于T细胞活化必不可少。T细胞缺乏泛醌-细胞色素C还原酶

(Uqcrfs1)，电子传递链的复合物III的一个组成部分，就显示受损的TCR依赖的活性氧产生和抗原特异性增殖的缺失(31)。细胞内的钙(Ca2+)流出，是TCR信号转导的早期事件，它可能提供TCR接合、线粒体氧化磷酸化及细胞增殖之间的功能性关系。线粒体摄取的Ca2+激活TCA循环中Ca2+依赖的脱氢酶，驱使线粒体在早期T细胞活化过程中通过OXPHOS产生NADH和ATP (32)。缺少细胞凋亡调节剂Bax和Bak的T细胞，显示细胞内动态平衡的缺陷，呈现Ca2+依赖的线粒体ROS产量和TCR激活后T细胞增殖的减少(33)。无Bax/Bak 的T细胞中Ca2+信号的修复，恢复线粒体ROS的产生和T细胞活化。因此，尽管在许多生物环境里是有毒的，线粒体依赖的ROS可能装备T细胞并许可完全的T细胞的活化。 Metabolic Signatures Vary with Differentiation State

代谢的识别标志随着分化状态变化

Fig. 1. T cell metabolism changes over the course of an immune response.T cells display distinct metabolic profiles depending on their state of activation. Na?ve T cells (TN, blue) are metabolically quiescent;they adopt a basal level of nutrient uptake and use OXPHOS as their primary pathway of ATP production. Upon immune challenge, TEFF (green) cells shift to a state of metabolic activation characterized by increased nutrient uptake, elevated glycolytic and glutaminolytic metabolism, biomass accumulation,and reduced mitochondrial SRC. TEFF cells preferentially use glycolysis over OXPHOS for ATP production. Transition to the TM(orange) stage is characterized by a quiescent metabolism, with increased reliance on FAO

to fuel OXPHOS. Mitochondrial mass and SRC are elevated in TM cells, suggesting that these cells are metabolically primed to respond upon reinfection.

图1.一个免疫应答的过程中T细胞代谢改变。T细胞基于它们的活化状态显示不同的代谢侧面。原始T细胞（TN，蓝色）在代谢上静止；它们采用基础水平的营养摄取并利用OXPHOS 作为它们产生ATP的主要途径。在免疫挑战时，TEFF (绿色)细胞转换为一个代谢活化的状态，特征是营养摄取增加、提高的糖酵解和谷氨酸代谢、生物物质积累、以及减少线粒体SRC。TEFF细胞优先通过OXPHOS利用糖酵解产生ATP。过渡到TM（橙色）阶段，以静止性代谢为特点，增加依赖FAO来为OXPHOS供能。在TM细胞中线粒体物质和SRC提高，提示这些细胞代谢上在再次感染时准备应答。

Although the paradigm of T cell metabolism as summarized in Fig. 1 holds true with respect to activated versus quiescent states, the metabolic signature of T cells can also vary depending on differentiation state. This was first demonstrated by Michalek et al.(34), who determined that proinflammatory CD4+T helper(TH) cells (TH 1, TH 2, and TH17 lineages) displayed a strong bias toward glycolysis over mitochondrial metabolism, whereas induced CD4+T regulatory (Treg) lineage cells displayed a mixed metabolism involving glycolysis, lipid oxidation, and OXPHOS.In particular, TH17 cells display increased reliance on glycolysis for their development and maintenance.TH17 cell development is promoted by hypoxia inducible factor–1α(HIF-1α)(35,36), an oxygen-sensitive transcription factor that regulates glycolytic gene expression in TH 17 cells.Blocking glycolysis during TH17 cell differentiation reduced the development of TH17 cells and favored the formation of Tregs (35). Added to this are recent results indicating that extracellular salt (NaCl) (37, 38) and short-chain fatty acids (39) can influence TH17 and Treg homeostasis, respectively.This raises the intriguing possibility that the metabolic microenvironment (i.e., nutrient and oxygen availability) can influence T cell polarization (to be discussed later). Determining whether the metabolic signature of differentiated T cells is simply a consequence of lineage-specific cytokine signaling or is instructive for T cell function (i.e., essential for regulating T cell plasticity and/or effector function) remains a question for the field. Examining the influence of key metabolic regulators such as HIF-1α, mTOR, and AMPK on T cell differentiation and plasticity will help in resolving these issues.

尽管如图1所总结的T细胞代谢范例相对于与静止比较的活化状态是正确的，但T细胞的代谢识别标志也会根据分化状态变化。这首先由Michalek等人所证明(34)，他们确定促炎症反应的CD4+T辅助（TH）细胞（TH1,TH2和TH17系）显示胜过线粒体代谢的对糖酵解的强烈偏好，而诱导的CD4+T调节(Treg)系的细胞显示涉及糖酵解、脂质氧化及氧化磷酸化的混合代谢。特别地，TH17细胞显示为了发展和维持对糖酵解依赖的增加。缺氧诱导因子—1α(HIF-1α)促进TH17细胞的发展(35,36)，它是调节TH17细胞中糖酵解基因表达的一种氧敏感的转录因子。对TH17细胞分化过程中糖酵解的阻碍，减少了TH17细胞的发展并促成Treg的形成(35)。补充这点的是，最近的结果提示细胞外的盐(NaCl) (37, 38)和短链脂肪酸(39)分别能影响TH17和Treg的动态平衡。这提高代谢微环境（即，营养和氧可利用性）能影响T细胞极化（将在稍后讨论）的有趣的可能性。确定已分化的T细胞的代谢识别标志仅仅是一系列系特异性的细胞因子信号转导，还是对T细胞功能有益（即，对调节T细胞可塑性和/或效应功能必不可少），在该领域仍是一个问题。检测例如HIF-1α、mTOR及AMPK等关键代谢调节剂对T细胞分化和可塑性的影响将有助于解决这些问题。

Metabolism of Proliferating Cells:Lessons from Tumor Metabolism

增殖细胞的代谢：来自肿瘤代谢的教训

Research in cancer metabolism over the past 10 years has increased our understanding of the metabolic requirements of proliferating cells, as well as the metabolic alterations that promote tumor growth. One of the great innovations we have learned from the cancer metabolism field is that signaling pathways, or more specifically the oncogenes and tumor suppressors that comprise and regulate signal transduction pathways, can influence cellular metabolism as part of their program of action. The previous paradigm of metabolic regulation argued that metabolic pathways were exclusively controlled through allosteric regulation of metabolic enzymes, either by ATP levels or metabolites themselves (the reactive model) (40). One example of this is the glycolytic enzyme phosphofructokinase (PFK), which is inhibited allosterically by ATP and citrate (indicating a high energy state in the cell) and stimulated by AMP (indicative of low energy). Although allosteric regulation is important for regulating local flux through metabolic pathways, we now understand that activation of signal transduction pathways (such as phosphatidylinositol 3-kinase or Akt) by growth factor receptors stimulates global changes in metabolic flux independent of ATP levels. This allows a cell that receives a proliferative signal, such as a T cell activated through its TCR, to drive cellular metabolism above the capacity normally maintained in the quiescent state. Overall, metabolism in T cells is likely regulated at several levels: (i) TCR-mediated changes in the expression of metabolic genes facilitate the reprogramming required to match metabolic pathways to biological need; (ii)signal transduction downstream of cell surface receptors (i.e., costimulatory molecules and cytokine receptors) serve to fine-tune flux through these metabolic pathways; and (iii) feedback inhibition and other forms of allosteric regulation can regulate metabolic flux through local nodes in the network. By directly influencing metabolic reprogramming, oncogenes and tumor suppressors gain control over the metabolic currency of the cell, namely, energetic intermediates (ATP, NAD+/NADH, FAD+/FADH2,and NADP+/NADPH) and metabolites involved in bioenergetic and biosynthetic reactions that influence cell growth and survival.

过去十年里肿瘤代谢的研究以及促进肿瘤生长的代谢改变已增加了我们对增殖细胞的代谢需求的理解。我们从肿瘤代谢领域学来的大的创新观念之一是，信号传导途径，或者更特异地，组成并调节信号转导途径的癌基因和肿瘤抑制物，能影响作为他们活动程序部分的细胞代谢。先前的代谢调节范例争论到，代谢途径通过代谢酶的变构调节被专门控制，经由ATP 水平或者代谢产物本身（活化型）(40)。其中一个例子是糖酵解酶磷酸果糖激酶(PFK)，PFK 被ATP和柠檬酸盐变构抑制（预示细胞内的高能状态）并被AMP激活（预示低能量）。尽管变构调节对调节代谢途径局部通量是重要的，但我们现在明白信号转导途径（例如磷脂酰肌醇3激酶或者Akt）通过生长因子受体，不依赖于ATP水平刺激代谢通量的整体改变。这允许一个接受增殖信号的细胞，例如通过自身TCR活化的一个T细胞，驱动优于在静止状态下正常维持的能力的细胞代谢。总之，T细胞内的代谢可能在几个水平调节：(i)TCR介导的代谢基因表达的改变，促进配合生物学需要的代谢途径要求的重编程；(ii)细胞表面受体（即，共刺激分子和细胞因子受体）的信号转导下游，有助于微调这些代谢途径的通量；并且(iii)反馈抑制和其它形式的变构调节能通过网络的局部结点调节代谢通量。靠直接影响代谢的重编程，癌基因和肿瘤抑制物加强控制细胞的代谢流通，即，能量中间体(ATP, NAD+/NADH, FAD+/FADH2和NADP+/NADPH)和涉及影响细胞生长生存的生物能学的与生物合成的反应的代谢产物。

The implication of these findings for immunologists is that metabolic pathways are indirectly

connected to cell surface receptors of the immune system via signal transduction pathways.TCR/CD28 stimulation of T cells (6), the stimulation of surface immunoglobulin on B cells (41),and TLR stimulation of macrophages and dendritic cells (DCs) (42,43) all promote changes in aerobic glycolysis characteristic of the Warburg effect. These results likely just scratch the surface of the complex metabolic networks at work in proliferating cells. The challenge going forward will be to identify key pathways of metabolic flux integral for lymphocyte function. In this regard,research into tumor cell metabolism has provided valuable insight into the metabolic pathways important for cell proliferation. Many of the metabolic pathways abnormally activated in cancer, such as aerobic glycolysis, have been shown to play similar roles in normal lymphocyte physiology. Here, we highlight recent advances in intermediate metabolism observed in cancer that are likely to be relevant to T cell biology. 这些发现给免疫学家的暗示是，代谢途径通过信号转导途径间接联系免疫系统的表面受体。T细胞的TCR/CD28刺激(6)、B细胞表面球蛋白的刺激(41)、以及巨噬细胞和树突状细胞的TLR刺激(DCs) (42,43)，都促进在有氧糖酵解Warburg效应特征方面的改变。这些结果可能只抓到在增殖细胞中起作用的复杂代谢网络的浅表。前进的挑战将是识别淋巴细胞功能所必须的代谢通量的关键途径。从这点看，肿瘤细胞代谢中的研究已经提供了对于细胞增殖重要代谢途径的有价值的洞见。许多在肿瘤中异常激活的代谢途径，例如有氧糖酵解，已被表明在正常的淋巴细胞生理中发挥类似作用。这里，我们强调在肿瘤中观察到的可能与T细胞生物学有关的中间代谢的最近进展。

The Warburg Effect: More than ATP Synthesis

Warburg效应：不仅是ATP合成

Rapid glucose processing promoted by the Warburg effect allows proliferating T cells to generate ATP quickly; glycolysis also generates metabolic intermediates important for cell growth and proliferation (Fig. 2). Metabolism of glucose through the oxidative or nonoxidative arms of the pentose phosphate pathway (PPP) generates ribose-5-phosphate (Rib-5P), a key intermediate in nucleotide biosynthesis. The oxidative arm of the PPP also produces NADPH, the key metabolic currency for nucleotide and fatty acid biosynthesis. T cell activation promotes a rapid increase in glucose flux through the oxidative PPP (9).Dihydroxyacetone-phosphate (DHAP) is used to generate the glycerol backbone for glycerophospholipids, and 3-phosphoglycerate (3PG) is a key intermediate in both amino acid and nucleotide biosynthesis (discussed below). Pyruvate that is not converted to lactate can enter into the mitochondria and be converted into acetyl-CoA by the pyruvate dehydrogenase (PDH) complex.In proliferating cells, mitochondria adopt an additional role as a biosynthetic hub, converting pyruvate and other metabolites into metabolic intermediates involved in protein and fatty acid biosynthesis (44).

由Warburg效应促进的快速葡萄糖处理允许增殖的T细胞很快产生ATP；糖酵解也产生对细胞生长和增殖重要的代谢中间物（图2）。通过磷酸戊糖途径(PPP)的氧化或非氧化的部分，葡萄糖代谢产生5-磷酸核糖(Rib-5P)，一种核苷酸生物合成的关键中间物。PPP氧化的部分（arm）也产生核苷酸和脂肪酸生物合成的关键代谢通货NADPH。通过氧化的PPP，T细胞活化促进葡萄糖通量的快速增加(9)。磷酸二羟丙酮(DHAP)被用来产生环甘油磷脂的甘油主链，而3-磷酸甘油酸(3PG)同时是氨基酸和核苷酸生物合成的关键中间物（下文讨论）。没被转化为乳酸的丙酮酸能进入线粒体并被丙酮酸脱氢酶(PDH)复合物转化为乙酰辅酶A。在增殖的细胞，线粒体采取作为一个生物合成枢纽的附加作用，将丙酮酸和其它代谢产物转化为参与蛋白及脂肪酸生物合成的代谢中间物(44)。

One of the key metabolic intermediates for biosynthesis is acetyl-CoA. Acetyl-CoA has a central role in membrane biogenesis because it provides two-carbon units for fatty acid and isoprenoid biosynthesis, as well as in other diverse processes such as protein prenylation and N-glycosylation (45). The flow of glucose to the cytosolic acetyl-CoA pool is regulated by using TCA cycle intermediates and a truncated TCA cycle (46). In this model, mitochondrial citrate is formed from condensation of oxaloacetate and acetyl-CoA, after which citrate is exported from the mitochondrion to the cytosol and converted back to acetyl-CoA by ATP citrate lyase(ACL) (47). Despite the availability of extracellular lipids for membrane biosynthesis, FFAs are generated de novo from glucose in proliferating cells using this pathway (46). CD8+ T cells unable to engage acetyl-CoA–dependent lipid biosynthetic pathways display defects in antigen-driven blastogenesis and clonal expansion in response to pathogens (48). Acetyl-CoA can also influence metabolic flux through acetylation of metabolic enzymes (49, 50), reinforcing metabolic pathways such as glycolysis when carbon availability is high. Glucose availability and acetylCoA production can also influence epigenetics by regulating the cytosolic acetyl-CoA pools available for histone acetylation reactions (51), raising the prospect that glucose-dependent metabolic flux may help drive or reinforce T cell differentiation programs.

生物合成的关键代谢中间物之一是乙酰辅酶A。乙酰辅酶A在膜生物合成中起中心作用，因为它为脂肪酸和类异戊二烯的生物合成提供两个碳单元，在例如蛋白质异戊烯化和N端糖基化的其它多种多样的加工中也一样(45)。TCA循环的中间物和截断的TCA循环，调节葡萄糖流向胞浆乙酰辅酶A池。该模式中，线粒体的柠檬酸盐形成于草酰乙酸和乙酰辅酶A的缩合，之后柠檬酸盐被从线粒体排出到胞质并被ATP柠檬酸裂解酶(ACL)转化回乙酰辅酶A (47)。细胞外的脂质除了对膜生物合成有用外，在增殖细胞中用此途径从头产生FFAs(46)。CD8+ T细胞不能进行依赖乙酰辅酶A的脂质生物合成途径，显示抗原驱动的母细胞化和对抗原应答的克隆扩增两方面的缺陷(48)。乙酰辅酶A也能通过代谢酶的乙酰化作用影响代谢通量(49, 50)，当碳利用率高时加强例如糖酵解的代谢途径。葡萄糖利用率和乙酰辅酶A的产量也能通过调节对组蛋白乙酰化反应有用的胞浆乙酰辅酶A池来影响表观遗传学(51)，提高依赖葡萄糖的代谢通量可能有助于驱动或加强T细胞分化程序的前景。

One consequence of using glucose-derived mitochondrial citrate for lipid biosynthesis is the potential depletion of TCA cycle intermediates.Oxaloacetate (OAA) is a rate-limiting substrate for acetyl-CoA entry into the TCA cycle. OAA generated from ACL-mediated cleavage of cytosolic citrate can potentially cycle back into the mitochondria to maintain the TCA cycle. Inefficient cycling of this pathway would lead to cumulative depletion of mitochondrial OAA,leading to collapse of the TCA cycle and disruption of mitochondrial function. One way tumor cells counter this is by engaging glutaminolysis, a metabolic shunt that converts glutamine into α-ketoglutarate (α-KG, also known as 2-oxoglutarate) for use in the TCA cycle (Fig. 2).Glutamine has long been known as a key metabolite for supporting T cell function (52). Recent evidence suggests that glutamine metabolism is as dynamically regulated in T cells as glucose metabolism. Glutamine transporters (SNAT1and SNAT2) as well as key glutaminolysis enzymes (GLS, GPT, GOT, and GLUD) are upregulated early after T cell activation similar to glycolysis genes (7, 9); several groups have correlated these changes in gene expression to enhanced glutaminolytic flux in lymphocytes (9,53).Recently, it was found that engagement of the TCR leads to the expression of Slc7a5, an amino acid transporter that mediates the import of large neutral amino acids, such as leucine (8). Amino acid influx via this transporter

is required for the activation of mTOR and expression of c-Myc and as such coordinates activation-induced metabolic reprogramming and differentiation of T cells.Thus, amino acids such as glutamine and leucine appear to play additional roles in T cell function beyond protein biosynthesis and may directly influence T cell activation by regulating metabolic reprogramming.

利用来自葡萄糖的线粒体柠檬酸进行脂质生物合成的一个后果是TCA循环中间物潜在的耗竭。草酰乙酸(OAA)是乙酰辅酶A进入TCA循环的一个速率限制性底物。产生于ACL介导的胞浆柠檬酸裂解的OAA，能潜在地循环回线粒体来维持TCA循环。该途径的无效循环将导致线粒体OAA的累积性耗竭，引起TCA循环的的崩溃和线粒体功能的中断。肿瘤细胞对付这个的一种方式是通过进行谷氨酰胺代谢的一种代谢分流，将谷氨酰胺转化为α-酮戊二酸盐（α-KG，也被称为2-酮戊二酸）用于TCA循环（图2）。谷氨酰胺长久以来就被认为是支持T细胞功能的关键代谢物(52)。最近的证据提示谷氨酰胺同葡萄糖代谢一样在T细胞中被动态地调节。谷氨酰胺转运蛋白(SNAT1和SNAT2)同关键的谷氨酰胺代谢酶(GLS, GPT, GOT和 GLUD)一样类似于糖酵解基因在T细胞活化后被上调(7, 9)；几个代谢组已经相互关联了在基因表达中的这些改变，来加强淋巴细胞中的谷氨酰胺通量(9,53)。最近发现，TCR的参与导致Slc7a5的表达，Slc7a5是介导例如亮氨酸的大的中性氨基酸输入的氨基酸转运体(8)。mTOR的活化和c-Myc的表达，以及与此协调的活化诱导的代谢重编程和T细胞分化，需要经由该转运体的氨基酸流入。这样，像谷氨酰胺和亮氨酸的氨基酸，显示出在蛋白生物合成之外的T细胞功能中的额外作用，并可能通过调节代谢重编程直接影响T细胞活化。 Control of Glycolytic Flux by Pyruvate Kinase M2 (PKM2)

丙酮酸激酶M2(PKM2)对糖酵解通量的控制

Fig. 2. Metabolic pathways that support cell growth and proliferation.Glycolysis and the TCA

cycle are two separate yet connected biochemical pathways that function to generate ATP as well as metabolic precursors for biosynthesis. Glucose is broken down to pyruvate by glycolysis (orange); pyruvate can be further oxidized by the TCA cycle in the mitochondrion. Glycolytic intermediates can be used to generate other metabolites required for growth and proliferation. Glucose 6-phosphate and 3PG produced from glycolysis are metabolized in the PPP (green) and the SBP (blue), respectively, providing important precursors for nucleotide biosynthesis. Similarly, acetyl-CoA, generated from glucose-derived citrate in the TCA cycle, can be used for lipid biosynthesis. OAA, produced as part of the TCA cycle, can be used to generate aspartate, another precursor for nucleotide synthesis. An alternate source of carbonfor the TCA cycle occurs via glutaminolysis (purple); in this pathway, glutamine is converted to glutamate and then toa-KG, which joins the TCA cycle. Glutamine is also a precursor for amino acid and nucleotide biosynthesis. Key enzymes in these pathways are PHGDH; PKM2;LDHA, lactate dehydrogenase; PDH; GLS, glutaminase; SDH, succinate dehydrogenase; and FH.

图2.支持细胞生长和增殖的代谢途径。糖酵解和TCA循环是两个独立又连接的生化途径，功能是产生ATP和生物合成的代谢前体。葡萄糖通过糖酵解（橙色）分解为丙酮酸；丙酮酸能在线粒体中通过TCA循环进一步被氧化。糖酵解中间物能被用于产生生长和增殖需要的其它代谢产物。产生于糖酵解的6-磷酸葡萄糖和3PG，分别在PPP（绿色）和SBP（蓝色）中被代谢，提供核苷酸生物合成的重要前体。类似地，乙酰辅酶A，产生于TCA循环中葡萄糖来源的柠檬酸，能被用于脂质生物合成。OAA，作为TCA循环的部分被产生，能被用于产生天冬氨酸，另一个核苷酸生物合成的前体。TCA循环的一个可选的碳来源通过谷氨酰胺代谢（紫色）发生；在这个途径，谷氨酰胺被转变为谷氨酸，然后变成α-KG，α-KG参与TCA循环。谷氨酰胺也是氨基酸和核苷酸生物合成的一个前体。这些途径中的关键酶是：PHGDH; PKM2;LDHA, 乳酸脱氢酶; PDH; GLS, 谷氨酰胺酶; SDH, 琥珀酸脱氢酶; 以及FH。

Pyruvate kinase (PK) is a key enzyme of the glycolytic pathway. It catalyzes the terminal reaction of glycolysis by promoting the conversion of phosphoenolpyruvate (PEP) to pyruvate(Fig. 2), and is one of two ATP-generating steps of glycolysis (the second is mediated by phosphoglycerate kinase). The muscle version of PK exists as one of two isoforms, M1 or M2, generated from differential splicing of the PKM primary mRNA transcript, with the PKM2 splice variant expressed in embryonic tissues, proliferating cells,and tumor cells (54). Na?ve T cells express both M1 and M2 isoforms at rest; mitogen-dependent activation promotes the rapid accumulation of the M2 isoform, which becomes the dominant isoform expressed in TEFF cells (55). PKM2 exists as either an inactive dimer or an active tetramer, and oscillation between these two states influences the ability of cells to maintain anabolic metabolism (56). 丙酮酸激酶(PK)是糖酵解途径的一个关键酶。它通过促进磷酸烯醇式丙酮酸(PEP)转化为丙酮酸，催化糖酵解的终末反应（图2），并且是糖酵解两个ATP生成步骤之一（另一个由磷酸甘油酸激酶介导）。肌肉中的PK以M1或M2两种异构体之一存在，产生于PKM初始mRNA 的不同剪接，同时PKM2的剪接变异体表达于胚胎组织、增殖的细胞及肿瘤细胞(54)。原始T细胞在静止时同时表达M1和M2异构体；依赖有丝分裂原的活化促进M2异构体的快速积累，变成TEFF细胞中表达的主要异构体(55)。PKM2以无活性的二聚体或者有活性的四聚体存在，而在这两种状态之间的波动影响细胞维持合成代谢的能力(56)。

Multiple lines of evidence point to a role for PKM2 in coordinating glycolytic flux and cell proliferation. Tumor cells engineered to exclusively express the M2 isoform display increased

lactate production characteristic of the Warburg effect and gain a growth advantage in vivo over M1-expressing tumors (57). Interestingly, the M2 isoform is actually less efficient at converting PEP to pyruvate than the M1 isoform. Moreover, growthfactor–stimulated tyrosine phosphorylation of PKM2 further decreases its activity (58,59). This prompts the question: Why would proliferating cells, including T cells, promote the expression of a pyruvate kinase isoform that is less efficient at generating ATP? The answer to this question may lie with the second function of Warburg metabolism, namely, supporting anabolic growth. Buildup of PEP because of reduced PKM2 activity promotes the accumulation of glycolytic intermediates, which can then be shunted into upstream biosynthetic pathways to support amino acid, triglyceride, and nucleotide biosynthesis.Activating PKM2 by using small-molecule agonists increases PEP-to-pyruvate conversion, reducing the flux of glycolytic intermediates toward anabolic pathways and slowing tumor cell growth(56,60). Thus, the ability of PKM2 to support cell proliferation may have less to do with ATP production and more with supporting biosynthetic pathways required for tumor cell growth.

多条线索的证据指明PKM2在协调糖酵解通量和细胞增殖中的作用。被设计专门表达M2异构体的肿瘤细胞显示高乳酸生产特点的Warburg效应，并获得胜过表达M1的肿瘤的体内生长优势(57)。有趣的是，M2异构体确实比M1异构体在将PEP转化为丙酮酸方面更低效。此外，生长因子刺激的PKM2酪氨酸磷酸化进一步降低它的活性 (58,59)。这引发一个问题：为什么增殖的细胞，包括T细胞，促进表达一种低效产生ATP的丙酮酸激酶异构体？这个问题的答案可能藏于Warburg代谢的第二个功能，即，支持合成代谢的生长。因为PKM2活性减低导致的PEP堆积，促进糖酵解中间物的积累，这然后能分流进入上游的生物合成途径来支持氨基酸、甘油三酯和核苷酸的生物合成。通过使用小分子激动剂活化的PKM2增加PEP向丙酮酸的转化，减少糖酵解中间物流向合成代谢途径并减慢肿瘤细胞生长(56,60)。因此，PKM2支持细胞增殖的能力可能与ATP生成的关系较少，而与支持肿瘤细胞生长需要的生物合成途径的关系较多。

PKM2 may also exert some of its effects on cell proliferation through nonmetabolic functions, including transcriptional and epigenetic regulation (61, 62). Of particular interest to immunologists is that PKM2 can phosphorylate signal transducer and activator of transcription 3 (STAT3)at Tyr705, promoting increased STAT3-dependent transcription (63). The phosphorylation of STAT3 by PKM2 demonstrates that PKM2 possesses both protein and pyruvate kinase activities, the former using PEP as a phosphate donor rather than ATP. The protein kinase activity of PKM2(and any subsequent effects on STAT3 activity)would be predicted to be sensitive to metabolic flux, favored under high-glycolysis PEP conditions and antagonized by low-energy high-ADP concentrations. The development of mouse models to study the impact of PKM2 activity in vivo will be important for elucidating the role(s) of PKM2 in immune function.

PKM2也可能通过非代谢的功能发挥它在细胞增殖的一些效应，包括调节转录和表观遗传学(61, 62)。使免疫学家特别感兴趣的是，PKM2能在第705位酪氨酸磷酸化信号转导和转录激活因子3 (STAT3)，促进依赖STAT3的转录增加(63)。STAT3被PKM2磷酸化显示，PKM2同时具有蛋白激酶和丙酮酸激酶的活性，前者利用PEP作为一个磷酸供体而不是利用ATP。PKM2的蛋白激酶活性（以及对STAT3活性的任何后续效应）可预测是对代谢通量敏感的，在高糖酵解PEP条件下被促进，而被低能量的高ADP条件拮抗。研究体内PKM2活性的影响的老鼠模型的发展，对阐明PKM2在免疫功能中的作用将是重要的。

The Serine Biosynthesis Pathway

丝氨酸生物合成途径

Fig. 3. Serine biosynthesis and reductive carboxylation are anabolic pathways that support cell proliferation.(A) The SBP converts glucose-derived 3PG into serine and glycine, which are precursors for lipid and nucleotide biosynthesis. Serine is also involved in folate-mediated one carbon metabolism by acting as a methyl group donor for THF to methylene-THF conversion. Key enzymes in this pathway are PHGDH, PSAT, and SHMT. (B) Reductive carboxylation is an alternate pathway of glutamine metabolism in which glutamine-derivedα-KG is converted to citrate through reverse TCA cycle flux. Under conditions of hypoxia or mitochondrial dysfunction (right), isocitrate dehydrogenase (IDH1 in cytosol, IDH2 in mitochondria) uses CO2 and NADPH to convertα-KG into isocitrate. Citrate produced downstream of this reaction is converted into cytosolic acetyl-CoA without passing through the conventional clockwise steps of the TCA cycle. Acetyl-CoA generated by this pathway can function as a precursor for fatty acid synthesis. α-KGDH, α–KG dehydrogenase.

图3，丝氨酸生物合成和还原性羧化作用是支持细胞增殖的合成代谢途径。（A）SBP转化葡萄糖衍生的3PG为丝氨酸和甘氨酸，它们是脂质和核苷酸生物合成的前体。丝氨酸也参与叶酸介导的一碳代谢，通过作为THF到亚甲基-THF的转变的一个甲基供体。在该途径关键的酶是PHGDH、PSAT和SHMT。（B）还原性羧化作用是谷氨酰胺代谢的一个交替的途径，其中谷氨酰胺来源的α-KG通过反向的TCA通量被转化为柠檬酸。在缺氧或线粒体功能障

碍（右）的条件下，异柠檬酸脱氢酶（IDH1在胞质，IDH2在线粒体）利用CO2和NADPH 转化α-KG为异柠檬酸。不用通过TCA循环常规的的顺向步骤，该反应下游产生的柠檬酸被转化为胞浆的乙酰辅酶A。由该途径产生的乙酰辅酶A能作为脂肪酸合成的一个前体。α-KGDH，α–KG 脱氢酶。

Another glycolytic intermediate that can double as an anabolic precursor is 3PG. 3PG is the starting point for the glucose-dependent biosynthesis of serine and glycine via the serine biosynthesis pathway (SBP) (Fig. 3A). Key enzymes of the SBP are phosphoglycerate dehydrogenase (PHGDH), the rate-limiting step for serine biosynthesis, and serine hydroxymethyltransferase (SHMT),which uses serine as a methyl donor to convert tetrahydrofolate (THF) to methylene-THF, generating glycine in the process. Methylene-THF is a key intermediate in folate-mediated one-carbon metabolism that fuels nucleotide biosynthesis and methylation reactions. Serine is also an allosteric activator of PKM2 (64) and thus provides feedback to the glycolytic pathway to regulate 3PG levels and serine biosynthesis.

能兼作一个合成代谢前体的另一个糖酵解中间物是3PG。3PG是经由丝氨酸生物合成途径(SBP)依赖葡萄糖的丝氨酸和甘氨酸生物合成的起始点(图3A)。SBP的关键酶是磷酸甘油酸脱氢酶(PHGDH) 和丝氨酸羟甲基转移酶(SHMT)，PHGDH是丝氨酸生物合成的限速步骤， SHMT利用丝氨酸作为甲基供体将四氢叶酸转化为亚甲基，在这过程中产生甘氨酸。亚甲基四氢叶酸是叶酸介导的一碳代谢的关键中间物，一碳代谢供能核苷酸生物合成和甲基化反应。丝氨酸也是PKM2的一个变构激活剂(64)，从而提供反馈给糖酵解途径来调节3PG水平和丝氨酸生物合成。

In mammals, serine and glycine are nonessential amino acids and are widely abundant in serum (and tissue culture medium). However,glucose-dependent serine biosynthesis is actively engaged in some tumors regardless of serine abundance. Amplification of PHGDH has been observed in breast cancer and melanoma (65),and increased PHGDH expression can promote both enhanced serine biosynthesis and the proliferation of cancer cells (65, 66). Enhanced flux through the SBP may confer a growth advantage to tumor cells beyond providing increased serine and glycine for biosynthetic reactions. First,PHGDH produces NADH, which can be used to maintain cytosolic redox balance or fuel mitochondrial OXPHOS to make ATP. The second step of the pathway, the conversion of 3-phosphohydroxypyruvate to 3-phosphoserine by phosphoserine aminotransferase (PSAT), requires glutamate and producesa-KG (Fig. 3A)(66). Thus, the SBP may promote an alternate pathway of α-KG production for mitochondrial metabolism or promote the activity of α-KG–dependent enzymes (to be discussed later). Last, serine and glycine are both intermediates in the production of reduced glutathione (GSH), a key cellular antioxidant. Recent evidence indicates that cancer cells actively produce GSH from glucose via the SBP as a buffer against oxidative damage (67). The expression of SBP enzymes is up-regulated in Myc-driven lymphomas (68); thus,we hypothesize that TEFF cells may actively engage the SBP upon activation, even in the presence of exogenous serine. Future investigation will be needed to determine how the SBP influences T cell biology.

在哺乳动物，丝氨酸和谷氨酸是非必需氨基酸，并且在血清中（以及组织培养基）是广泛丰富的。不管怎样，葡萄糖依赖性丝氨酸生物合成积极参与一些肿瘤，无论丝氨酸的丰度。PHGDH的放大作用被观察于乳腺癌和黑色素瘤(65)，并且PHGDH表达的增加能同时促进

丝氨酸生物合成增强和肿瘤细胞的增殖(65, 66)。通过SBP增高通量，除了为生物合成反应提供增加的丝氨酸和谷氨酸之外，可能赋予肿瘤细胞生长优势。首先，PHGDH 产生 NADH，NADH能被用于维持胞浆的氧化还原的平衡或者为线粒体氧化磷酸化供能来制造ATP。该途径的第二个步骤，通过磷酸丝氨酸转氨酶(PSAT)将3-磷酸羟基丙酮酸转化为3-磷酸丝氨酸，需要谷氨酸并产生α-KG（图3A）(66)。因此，SBP可能为线粒体代谢促进α-KG生成的代替途径，或者促进依赖α-KG的酶的活性（后面将讨论）。最后，丝氨酸和甘氨酸都是还原型谷胱甘肽(GSH)产生过程的中间物，GSH是一个关键的细胞抗氧化剂。最近证据提示肿瘤细胞经由SBP积极从葡萄糖产生GSH，作为抗氧化损伤的一个缓冲剂(67)。在Myc基因诱导的淋巴瘤中SBP酶的表达被上调(68)；因此，我们假设TEFF细胞在活化时可能积极进行SBP，甚至外源性丝氨酸存在时。将需要未来的研究来确定SBP怎样影响T细胞生物学。 Reductive Carboxylation ofα-KG

α-KG的还原性羧化

As mentioned previously, it is now appreciated that in proliferating cells the TCA cycle functions as a source of biosynthetic precursors in addition to its role in ATP production (46). Carbon enters the TCA cycle primarily at one of two entry points:(i) the condensation of glucose-derived acetyl-CoA with OAA to generate citrate and (ii) conversion of glutamine to α-KG via glutaminolysis(Fig. 3B). Carbon-tracing experiments using proliferating glioblastoma cells have established that both glucose and glutamine contribute to mitochondrial citrate pools and subsequent lipid synthesis (69). The oxidative decarboxylation of isocitrate toα-KG by isocitrate dehydrogenase(IDH) is highly favored thermodynamically, such that this reaction is believed to be irreversible and the reason for the“clockwise”flow of metabolic intermediates through the TCA cycle.

如先前提到的，现在赏识，在增殖的细胞TCA循环除了产生ATP，还有生物合成前体的来源的功能(46)。碳主要在两个切入点之一进入TCA循环：(i)来自葡萄糖的乙酰辅酶A和OAA 的缩合产生柠檬酸，以及(ii)谷氨酸通过谷氨酰胺转变为α-KG（图3B）。使用增殖的胶质母细胞瘤细胞的碳追踪实验已经证实，葡萄糖和谷氨酸都有助于线粒体柠檬酸池和随后的脂质合成(69)。异柠檬酸通过异柠檬酸脱氢酶(IDH)氧化脱羧为α-KG被热力学地高度促进，这样该反应被相信是不可逆的并且是代谢中间物通过TCA循环“顺时针” 单向流动的原因。 Groundbreaking new work suggests that metabolite flow through biochemical pathways does not always conform to conventional dogma.Although oxidation of glutamine-derivedα-KG in the TCA cycle serves as a minor source of lipogenic acetyl-CoA under normal growth conditions, α-KG can be converted to citrate through reductive carboxylation under conditions of stress such as hypoxia or mitochondrial dysfunction (70–72). In this reaction, glutamine-derivedα-KG is carboxylated, rather than decarboxylated, by IDH1 (in the cytosol) or IDH2 (in mitochondria) to generate citrate (Fig. 3B). This switch to reductive versus oxidative metabolism of α-KG is regulated in part by HIF-1α(71,72), although the exact mechanism by which HIF-1αdoes so remains unclear. Engaging reductive carboxylation of α-KG in essence bypasses the conventional TCA cycle by using glutamine to generate the acetyl-CoA required for fatty acid synthesis (Fig. 3B). Under such metabolic reprogramming, cancer cells continue to use glycolysis for ATP production but switch from glucose to glutamine as the major lipogenic precursor.

突破性的新工作提示，经过生化途径的代谢流不是经常符合传统的教条。尽管在正常生长条件下来自谷氨酸的α-KG的氧化作为脂源性乙酰辅酶A的小来源，在应激条件下例如缺氧或者线粒体功能障碍，α-KG能通过还原羧化作用转变为柠檬酸(70–72)。在这个反应，谷氨酸

来源的α-KG被羧化，而不是被脱羧，通过IDH1 (在胞质) 或者IDH2 (在线粒体)来生成柠檬酸（图3B）。α-KG还原性与氧化性代谢的转换被HIF-1α(71,72)部分调节，虽然HIF-1α靠什么这样做的确切的机制还不清楚。α-KG吸引人的还原性羧化作用在本质上绕过传统的TCA循环，通过利用谷氨酸来生成脂肪酸合成所需的乙酰辅酶A（图3B）。在这样的代谢重编程下，肿瘤细胞继续利用糖酵解产生ATP，但从葡萄糖转向将谷氨酸作为主要的脂源性前体。

One implication of this work is that tumor cells display a high degree of metabolic plasticity and can adapt their metabolism to support proliferation and viability under fluctuating environmental conditions.Whether T cells display similar metabolic flexibility in response to environmental cues requires further investigation. The work from Metallo and colleagues (71) suggests that T cells can use reductive glutamine metabolism for fatty acid biosynthesis under hypoxic conditions.Physiologic oxygen tension varies significantly between tissues, and hypoxic regions can be detected in the bone marrow, thymus, and spleen(73). Moreover, T cells are likely to experience hypoxia at sites of tissue inflammation (74). Stabilization of HIF-1α and metabolic reprogramming to favor reductive carboxylation may help T cells maintain proliferation and/or effector function at hypoxic inflammatory sites. HIF-1α protein expression has also been observed early after T cell activation (75), so it is unclear whether reductive carboxylation may also be engaged as part of the normal program of T cell expansion.As mentioned, HIF-1α has been implicated in the differentiation of both proinflammatory TH17 cells (35,36) and CD4+FoxP3+Tregcells (76). It is tempting to speculate that this alternate pathway of glutamine metabolism may influence the expansion or phenotypic stability of these T cell subsets.

这项工作的暗示是，肿瘤细胞显示高度的代谢可塑性并能使它们的代谢适应在波动的环境条件下支持增殖和生存能力。T细胞对环境信号应答时是否显示类似的代谢灵活性，需要进一步的研究。来自Metallo和同事们的工作（71）表明T细胞能利用还原性谷氨酸代谢在缺氧条件下合成脂肪酸。生理氧张力在组织间有意义地变化，并且在骨髓、胸腺以及脾能检测到缺氧区域(73)。此外，T细胞可能在组织炎症位置经历缺氧(74)。HIF-1α的稳定性与促进还原性羧化的代谢重编程可能帮助T细胞在缺氧的炎症部位维持增殖和/或效应细胞作用。在T细胞活化后的早期也观察到HIF-1α蛋白表达(75)，所以不清楚是否还原性羧化可能也作为一部分参与T细胞扩增的正常程序。如提到的，HIF-1α被密切关联于促炎症反应的TH17细胞(35,36)和CD4+FoxP3+Treg细胞(76)的分化。吸引人的推测是，这个谷氨酸代谢的替代途径影响这些T细胞亚群的扩增或者表型稳定性。

Metabolites As Signaling Molecules

作为信号分子的代谢物

Cancer genome sequencing efforts yielded an unexpected discovery in 2008 with the identification of somatic mutations in a metabolic enzyme,the TCA cycle enzyme IDH1, in glioblastoma multiforme (GBM) (77). Mutations in IDH1 presented at significant frequency (~12% of GBM patients) with high frequency of missense mutations targeting an arginine residue in the active enzyme site (Arg132). This mutation alters the enzymatic ability of IDH1, allowing it to convert α-KG to 2-hydroxyglutarate (2-HG) rather than promote normal isocitrate–to–α-KG interconversion (78). In the cancer setting, 2-HG has been shown to influence a number of biological processes, including cell differentiation, DNA methylation, and histone methylation (79–82),leading to its classification as an oncometabolite. These results have shifted thinking in cancer biology to consider that specific metabolites may also

act as signaling molecules to influence cell physiology. Given certain similarities between metabolic programming in tumor cells and proliferating T cells, it stands to reason that metabolites will participate in T cell signaling as well.Some examples of how metabolites shape T cell function and fate through metabolic pathways are discussed in detail here.

在2008年，肿瘤基因组测序的努力产生了一个出乎意料的发现，即，在多形性胶质母细胞瘤(GBM)中TCA循环一个代谢酶IDH1的体细胞突变的鉴定(77)。在IDH1的突变以有意义的频率（~12%的GBM患者）出现，伴随在活性酶位点(Arg132)针对精氨酸残基的高频率错义突变。这个突变改变了IDH1的酶能力，使它转化α-KG为2-羟基戊二酸(2-HG)，而不是促进正常的异柠檬酸到α-KG的互变(78)。在肿瘤环境里，2-HG已显示影响许多生物过程，包括细胞分化、DNA甲基化及组蛋白甲基化(79–82)，导致它被归类为一个癌代谢物。这些结果已经转变在肿瘤生物学的想法去思考，特殊的代谢产物可能也作为信号分子去影响细胞生理。基于所给的肿瘤细胞和增殖的T细胞代谢重编程之间的相似性，按理说代谢产物将也参与T细胞的信号传导。代谢产物怎样通过代谢途径形成T细胞功能和命运的一些例子在这里具体讨论。

Regulation of LKB1-AMPK Signaling by Adenylates

靠腺苷酸的LKB1-AMPK信号调节

Fig. 4. Metabolites can influence signal transduction.(A) The AMPK pathway is influenced by adenylate concentration. AMPKα is activated by phosphorylation on Thr172 of its activation loop by the kinases LKB1, TAK1, or CaMKKβ.LKB1 promotes enhanced AMPK phosphorylation under a high AMP:ATP ratio. One biological output of AMPK activity is the inhibition of mRNA translation under low-energy conditions through inhibition of mTORC1 activity. (B) α-KG–dependent enzymes (in yellow) are a class of enzymes regulated by TCA cycle intermediates that require molecular oxygen (O2)and α-KG for their enzymatic activity. Oxygen, α-KG, ascorbate, and iron (green)are positive regulators of these enzymes, whereas the TCA cycle intermediates fumarate and succinate (blue) antagonize their reactions. PHDs destabilize HIF-1α protein, resulting in decreased expression of HIF-1α targets and a reduction in glycolysis. TET2 hydroxylates 5-methylcytosine residues to promote DNA demethylation, whereas JmjC promotes demethylation of trimethylated histones in chromatin.

图4. 代谢产物能影响信号转导。（A）AMPK途径受腺苷酸浓度影响。AMPKα靠它活性环的Thr172磷酸化被激活，通过激酶LKB1、TAK1或 CaMKKβ。在一个高AMP:ATP 比率下LKB1促进AMPK磷酸化增强。AMPK活性的一个生物学输出是，在低能量条件下mRNA翻译的

抑制，通过mTORC1活性的抑制。（B）α–KG依赖性酶（黄色的）是由TCA循环中间物调节的一类酶，它们的酶活性需要分子氧（O2）和α–KG。氧气、α–KG、抗坏血酸和铁绿色）是这些酶的积极调控者，然而TCA循环中间物延胡索酸和琥珀酸（蓝色）对抗它们的反应。PHDs使HIF-1α蛋白不稳定，导致HIF-1α目标的表达降低和糖酵解的减少。TET2羟基化5-甲基胞嘧啶残基来促进DNA去甲基化，然而JmjC促进染色质中三甲基化的组蛋白去甲基化。

ATP is the primary carrier of chemical energy in the cell and essential for life. Thus, adenylates[ATP, adeonsine diphosphate (ADP), and AMP]are important units of cellular metabolic currency. In mammalian cells, fluctuations in cellular energy are monitored by the heterotrimeric AMPK complex [reviewed in (83)]. ATP, ADP,and AMP compete for nucleotide-binding sites of the γ regulatory subunit of AMPK, with AMP (low energy) promoting and ATP (high energy) antagonizing AMPK activation. As such, AMPK functions as a sensor of the cellular adenylate energy charge (84, 85). Elevation of the cellular AMP:ATP ratio leads to increased phosphorylation of AMPK at Thr172 of its activation loop by the kinase LKB1 (Fig. 4A). AMPK can also be activated by Ca2+(via CamKKβ) and the cytokine transforming growth factor–β(TGF-β)(via TAK1), although LKB1 appears to be the sole kinase that couples adenylate binding to AMPK activation.

ATP是细胞中主要的化学能量载体并且对生命必不可少。因此，腺苷酸[ATP，二磷酸腺苷(ADP)和AMP]是细胞代谢的重要货币单位。在哺乳动物的细胞，细胞能量的波动被异三聚体的AMPK复合物监控[综述于(83)]。ATP、ADP和 AMP竞争AMPK的γ调节亚基的核苷酸结合位点，AMP（低能量）促进而ATP（高能量）拮抗AMPK活化。如此，AMPK作为细胞的腺苷酸能量指示的一个传感器起作用(84, 85)。细胞的AMP：ATP比例升高导致AMPK在Thr172的活化环磷酸化增加，通过激酶LKB1（图4A）。AMPK也能被Ca2+(通过CamKKβ)和细胞因子转化生长因子-β(TGF-β)(通过TAK1)活化，尽管LKB1似乎是连接绑定于AMPK活化的腺苷酸的唯一酶。

Together LKB1 and AMPK function as part of an evolutionarily conserved energy-sensing pathway that maintains cellular energy balance by promoting catabolic pathways of ATP production and limiting processes that consume ATP. Protein synthesis is one of the most energy consuming processes in the cell, accounting for ~20% of cellular ATP consumption (86). As mentioned, AMPK antagonizes mRNA translation through negative regulation of mTORC1 (Fig. 4A)(18). By regulating AMPK activity, adenylates directly influence pathways of energy usage in the cell. As ATP levels drop, AMP binds to AMPK, and AMPK is switched on to promote ATP production and block its consumption; once ATP homeostasis has been reestablished, increased binding of ATP to AMPK shuts off the kinase.

LKB1 和AMPK一起作为一个进化上保守的能量感知途径的一部分起作用，该途径通过促进ATP生成的分解代谢途径和限制消耗ATP的过程维持细胞的能量平衡。蛋白质合成是细胞中最耗能的过程之一，占~20%的细胞ATP消耗(86)。如提到的，AMPK通过mTORC1的负性调节拮抗mRNA翻译(图4A)(18)。通过调节AMPK活化，腺苷酸直接影响细胞内的能量利用途径。当ATP水平下降时，AMP结合到AMPK，而AMPK开始促进ATP产生并阻断它的消耗；一旦ATP动态平衡被重新建立，增加的结合到AMPK的ATP关闭该激酶。

Recent work indicates that LKB1-AMPK signaling can influence T cell metabolism and function. Lymphocytes exclusively express the α1 catalytic subunit of AMPK (87), and TCR stimulation promotes LKB1-dependent AMPK activation in lymphocytes (87, 88). Glycolysis is enhanced

in resting AMPKα-deficient T cells(88), consistent with observations that silencing AMPK in tumor cells promotes the Warburg effect (89). Loss of LKB1-AMPK signaling promotes increased mTORC1 activity in both na?ve and TEFF cells (88, 90), which in turn facilitates production of the TH1 cytokine interferon-γ(IFN-γ) by TEFF cells (88). Thus, LKB1 and AMPK act in concert to limit the anabolic growth of T cells by suppressing glycolysis and mTOR-dependent biosynthesis. Perhaps not surprisingly, deletion of either LKB1 or AMPKα1 disrupts normal lymphocyte homeostasis, resulting in an accumulation of activated (CD44hiCD62Llo)CD8+T cells in animals (88). These results suggest that cellular adenylate levels and AMPK may help regulate lymphocyte pools in the whole organism.

最近的工作表明LKB1-AMPK信号能影响T细胞代谢和功能。淋巴细胞专门表达AMPK的α1催化亚基(87)，并且TCR刺激促进淋巴细胞中依赖LKB1的 AMPK活化(87, 88)。在静止的AMPKα-缺失的T细胞中糖酵解被增强(88)，与观察到的肿瘤细胞中AMPK沉默促进效应一致(89)。LKB1-AMPK信号丢失促进原始的和TEFF细胞中mTORC1活化都增加(88, 90)，这转而促进TEFF细胞产生TH1细胞因子白介素-γ(IFN-γ) (88)。因此，LKB1 和AMPK作用符合限制T细胞的合成代谢生长，通过抑制糖酵解和依赖mTOR的生物合成。可能不奇怪，LKB1或者AMPKα1的消除扰乱正常的淋巴细胞动态平衡，导致动物中活化的(CD44hiCD62Llo)CD8+T细胞累积(88)。这些结果提示细胞的腺苷酸水平和AMPK可能帮助调节整个器官中的淋巴细胞池。

Why would a signaling pathway that normally monitors cellular energy levels regulate T cell function? Similar to tumor cells (91), AMPK may regulate a metabolic checkpoint in T cells, acting as a brake on lymphocyte expansion when energy conditions are poor. TEFF cells with defective AMPK signaling would be freed from these metabolic checkpoints and continue to proliferate and produce cytokines as if cellular bioenergetics were suitable to support T cell function. Additionally, AMPK may regulate the metabolic plasticity of lymphocytes, coordinating metabolic changes in response to nutrient fluctuation and allowing T cells to survive changes in their environment. As evidence for this, the AMPK agonist metformin, which promotes FAO in activated T cells, enhances the production of CD8+ memory T cells in vivo (17). Furthermore, it was recently shown that AMPK-deficient T cells are defective in their ability to generate CD8+ memory T cells during infection (92). There is also a growing body of evidence implicating AMPK in the control of inflammation (3). Future work will focus on the role of AMPK in regulating the metabolic fitness of lymphocytes, dissecting LKB1-and AMPK-specific effects on immune function and investigating the role(s) of LKB1 and AMPK in regulating inflammation in vivo.

为什么一个正常地监控细胞能量水平的信号途径可调节细胞的功能？类似于肿瘤细胞(91)，AMPK可能调节T细胞中的一个代谢关卡，当能量状况差时作为淋巴细胞扩增的一个闸门起作用。缺失AMPK信号的TEFF细胞可摆脱这些代谢的关卡并继续增殖和产生细胞因子，好像细胞的生物能量适合支持T细胞功能。此外，AMPK可能调节淋巴细胞的代谢可塑性，协调对营养波动反应的代谢变化并允许T细胞在它们的环境变化中幸存。作为这的证据，AMPK 的激动剂二甲双胍，促进活化的T细胞中的FAO，增强体内CD8+记忆T细胞的产生(17)。再者，最近表明AMPK缺陷的T细胞在感染过程中产生CD8+记忆T细胞能力的缺陷(92)。在炎症控制方面越来越多的证据牵涉AMPK (3)。未来的工作将集中于AMPK在调节淋巴细胞的代谢适合度中的作用，剖析对免疫功能LKB1-和 AMPK-特异的作用并研究体内LKB1 和 AMPK调节炎症的作用。

Regulation of α-Ketoglutarate-Dependent Enzymes by TCA Cycle Metabolites