mtor signalling and metabolic regulation of t cell differentiation

Available online at www.sciencedirect.com

mTOR signalling and metabolic regulation of T cell differentiationChristian Peter, Herman Waldmann and Stephen P Cobbold

T cells constantly monitor energy status and nutrient levels in

order to adjust metabolic pathways according to their

nutritional status and other environmental stimuli. It is

increasingly evident that the regulation of cellular metabolism is

tightly coupled to T cell differentiation that ultimately

determines the cellular fate. The mammalian target of

Rapamycin (mTOR) pathway has emerged as a key player in

sensing these nutritional/energetic signals and in addition, acts

as a major integrator of growth factor induced signals, so

placing mTOR at the core of a signalling network controlling

metabolism and cellular fate. The mTOR pathway has been

shown to play an important role in determining the

differentiation of CD4+ T cells into inflammatory and regulatory

subsets, in the induction of anergy, in the development of CD8+

memory T cells and the regulation of T cell trafficking.

Address

Sir William Dunn School of Pathology, Oxford University, South Parks

Road, Oxford OX1 3RE, United Kingdom

Corresponding author: Waldmann, Herman

([email protected])

Current Opinion in Immunology 2010, 22:655–661

This review comes from a themed issue on

Immunogenetics and transplantation

Edited by Terry Strom and Allan D. Kirk

Available online 15th September 2010

0952-7915/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.coi.2010.08.010

T cell activation and mTOR regulatedmetabolic changesT cell activation is intimately linked to changes in the

mode of energy generation, providing the basis for the

energy demanding processes concurrent with T cell

differentiation. Whereas resting T cells generate most

of their energy by oxidative phosphorylation, T cell

activation is characterized by a marked increase in gly-

colysis [1]. This mode of energy generation is termed

‘aerobic glycolysis’ since it occurs despite a seemingly

adequate oxygen supply. What causes this switch remains

unclear. Based on the finding that increased mTOR

activity can stimulate oxygen consumption by direct

interaction with mitochondria or through an increased

energetic load caused, for example, by increased protein

synthesis, one could speculate that mTOR is involved in

these metabolic changes [2,3].

www.sciencedirect.com

In order to generate vast amounts of ATP from glucose

and provide sufficient amino acids (AA) and lipids for

protein and membrane synthesis respectively, T cells

have to co-ordinately and rapidly up-regulate import of

these reactants. In the case of glucose this might be

achieved through AMP-activated protein kinase (AMPK)

activation initiated by T cell receptor engagement and via

Ca2+/calmodulin-dependent kinase kinase (CAMKK2)

[4,5]. This might represent a mechanism by which T

cells can anticipate rising energy demands required by T

cell activation. Concurrently, activated T cells up-

regulate surface expression of glucose transporter Glut1

by increasing its trafficking to meet the increasing

demand for glucose. The rise in surface localisation of

Glut1 seems to be dependent on PI3K/Akt activation

resulting from TCR signalling and CD28 costimulation

[6]. Glucose uptake seems to be enhanced further

through mTOR-mediated activation of Glut1 [7].

In addition to increased glucose consumption activated

T cells require enhanced protein synthesis and other

anabolic pathways to facilitate cell growth, division and

differentiation, thereby necessitating enhanced uptake

of nutrients. T cell receptor and CD28 engagement

induce Akt signalling via mTOR which leads to higher

surface expression of a variety of nutrient transporters

and receptors including AA transporters and the trans-

ferrin receptor [8]. The fact that mTOR was originally

discovered in response to the development of rapamy-

cin as an immunosuppressive agent [9] highlights the

crucial importance of the mTOR signalling pathway in

T cell mediated immune responses, and further

suggests that nutrient availability and metabolic regu-

lation are intimately linked to T cell activation and

differentiation.

The sensing of nutrional and energeticstatus by mTORmTOR signalling, as a function of the TORC1 complex

[10�], has been implicated as a major integrator of signals

related to cellular nutrient and energy status. The mol-

ecular mechanisms by which TORC1 controls the nutri-

tional status of cells have only begun to be understood. In

the first instance, cell intrinsic energy availability is

sensed by AMPK, which is activated by rising AMP/

ATP ratios and hypoxia. AMPK negatively regulates

TORC1 activity through activation of the TSC1/2 com-

plex. This AMPK mediated sensing mechanism enables

TORC1 to adjust metabolic pathways and other vital

energy consuming cellular processes, such as protein

synthesis with respect to the cellular energy status

(Figure 1).


mailto:[email protected]

http://dx.doi.org/10.1016/j.coi.2010.08.010

656 Immunogenetics and transplantation

Figure 1

The mTORC1 signalling pathway. The serine/threonine protein kinase mTOR forms two structurally and functionally distinct complexes TORC1 and

TORC2. TORC1, the Rapamycin sensitive branch of the mTOR pathway, consists of the scaffolding protein Raptor, FKBP12 (the molecular target of

Rapamycin) and three further subunits, mLST8, PRAS40 and Deptor. TORC1 activity is critically controlled by a small GTPase, Rheb, whose activity is

inhibited by a GTPase activating protein, TSC2 in complex with TSC1. Consequently, inactivation of the TSC1/2 complex by upstream signals

emerging from the PI3K/Akt axis and the Erk pathway activate TORC1. Many accessory molecules as well as growth factors signal via the PI3K/Akt

and Erk pathways and therefore inevitably activate TORC1. Akt can further promote TORC1 activity independent of TSC1/2 by phosphorylating

PRAS40, a negative regulator of TORC1 activity. Furthermore TORC1 can be activated via the WNT pathway, in a signalling cascade involving GSK3.

Energy deficiencies as well as low oxygen levels lead to the activation of AMPK, which phosphorylates and activates the TSC1/2 complex resulting in

TORC1 inhibition. Furthermore, TORC1 activity is dependent on sufficient levels of amino acids, in a process involving recently discovered Rag A–D

proteins and the Ragulator complex. Active TORC1 controls a variety of cellular processes ranging from autophagy, lipid synthesis and cell cycle

progression to mitochondrial biogenesis and protein synthesis. The latter is at least in part controlled by the TORC1 downstream targets 4E-BP1 and

S6K1. Abbreviations: mTOR, mammalian target of Rapamycin; TORC, mTOR complex; Raptor, regulatory-associated companion of mTOR; FKBP12,

FK506 binding protein 12; mLST8, mammalian lethal with Sec13 protein 8; PRAS40, prolin-rich Akt substrate 40kDa; Deptor, DEP-domain-containing

mTOR-interacting protein; Rheb, Ras homologue enriched in brain; TSC, tuberous sclerosis protein; PI3K, Phosphatidylinositol 3-kinase; Akt, AKT8

virus oncogene cellular homologue; Erk, Extracellular signal-regulated kinase; GSK3, Glycogen synthase kinase 3; AMPK, AMP-activated protein

kinase; Rag A–D, Ras-related GTP-binding-proteins A–D; (EIF)4E-BP1, Eukaryotic translation initiation factor 4E-binding protein 1; S6K1, (ribosomal)

S6 kinase 1.

A second key nutrient sensed by TORC1 is the avail-

ability of AA. This component of TORC1 activity

requires a number of recently identified proteins (the

Ras-related GTP-binding-proteins A–D; Rag A–D) that

control the intracellular localisation and thereby the acti-

vation of TORC1 (Figure 2) [11�,12�]. These Rag

proteins form heterodimers that interact with TORC1


via raptor when the RagB component of the RagB/D

complex is in its GTP-bound form. AA induce the loading

of RagB with GTP and thereby facilitate the interaction

of the Rag complex with TORC1. Recently an additional,

trimeric protein complex called Ragulator was identified

and shown to interact with TORC1 and the Rag

GTPases. Comprising the MAPKSP1, ROBLD3, and


mTOR signalling and metabolic regulation of T cell differentiation Peter, Waldmann and Cobbold 657

Figure 2

Putative model for the amino acid mediated regulation of TORC1 activation. Amino acid induced translocation of mTOR is thought to regulate its

activity, in a process that involves the Rag A–D and the trimeric Ragulator complex. Amino acids facilitate loading of Rag B with GTP, thereby

permitting the binding of the Rag proteins to Raptor in TORC1. In conjunction with the Ragulator complex the Rag proteins are essential for the

movement of TORC1 to Rhab7+ lysosmal compartments, in which the TORC1 activator Rheb resides. Abbreviations: Rag A–D, Ras-related GTP-

binding-proteins A–D; mTOR, mammalian target of Rapamycin; Rheb, Ras homologue enriched in brain

c11orf59 gene products, Ragulator was demonstrated to be

essential for the localisation of TORC1 and the Rag

complex to a Rab7+ lysosomal compartment containing

the crucial TORC1 activator Rheb [13�]. Despite recent

advances in the understanding of TORC1 activation by

AA, the molecular sensor of AA within or outside the cells

has yet to be identified, although recent studies in Dro-

sophila have revealed a potential role of proton-assisted

amino acid-transporters (PAT) in sensing and transmit-

ting an AA signal to TORC1 [14,15]. Considering the fact

that TORC1 can regulate the activity of AA transporters

this model also might allow TORC1 signals to feed back

on its sensors [16,17].

mTOR and T cell differentiationThe differentiation of T cells is a highly complex process

initiated by multiple signals emerging from the TCR,

cytokine receptors and co-stimulatory molecules. These

signals and the microenvironment in which a T cell is

activated direct its fate towards the ever growing number

of functionally distinct T cell subsets. By integrating the

nutritional and energetic status of the cells into the

signalling events induced by T cell activation in the local

environment, the mTOR complexes allow the cells to


asses the wide spectrum of environmental stimuli as a

whole, so impacting the outcome of the differentiation

process.

It is widely accepted that the balance between effector T

cells and regulatory T cells is critical to the resulting

immune response. Studies performed by Delgoffe and

colleagues highlight the importance of mTOR in con-

trolling this delicate and important balance. Despite

displaying normal up-regulation of activation markers

and IL-2 production, T cells deficient in mTOR failed

to differentiate into inflammatory Th1, Th2 and Th17

subsets when activated. Furthermore the same activation

conditions led to the differentiation of Foxp3+ regulatory

T cells, indicating the importance of the mTOR pathway

in skewing CD4+ helper T cell fate [18��]. A recent study

utilizing Rictor knockout T cells demonstrates that the

differentiation of Th1 and Th2 is highly dependent on

TORC2 signalling leading to activation of Akt and PKC-u

respectively, whereas the development of Th17 cells

seems to be unimpaired by the lack of TORC2 activity

[19��]. Along these lines Kopf et al. confirmed that Rapa-

mycin prevented the differentiation of Th17 whereas

Foxp3 induction was promoted [20]. This role of the



mTOR pathway in promoting regulatory T cell differen-

tiation is underlined by experiments demonstrating that

components upstream of TORC1, such as PI3K and Akt

as well as TORC1 itself, negatively regulate Foxp3

expression [21�,22�]. Although these results suggest that

TORC1 inhibition is the crucial component allowing

Foxp3 to be induced, a number of caveats hinder a

definite conclusion. Although both studies indicate the

involvement of the mTOR pathway by demonstrating

that the observed effect is, for the most part, sensitive to

Rapamycin, neither group compared TORC1 or TORC2

activity. As the two complexes are thought to be recipro-

cally regulated and yet prolonged treatment with the

allosteric TORC1 inhibitor Rapamycin may result in

TORC2 inhibition, a straight forward analysis seems

difficult [23]. The interpretation of the findings of Hax-

hinasto et al. is further complicated given that Akt is also a

downstream target of TORC2, which might explain why

Rapamycin could not fully rescue the negative effect of

constitutively active Akt on Foxp3 induction [21�]. Along

these lines, Delgoffe et al. demonstrate that TORC2

inhibition might have a more important contribution to

the induction than previously assumed. Based on the

finding that T cells devoid of the TORC1 activator Rheb

failed to express Foxp3 under conditions that induced the

transcription factor in mTOR�/� T cells, the authors

postulated that TORC2 inhibition might be crucial in

facilitating Foxp3 induction by releasing Smad3 from an

inhibitory phosphorylation mediated via the TORC2

target Akt [18��].

We have demonstrated that TORC1 inhibition, by star-

ving T cells of essential AA, represents a physiological

mechanism that synergizes with TCR and TGFb sig-

nalling to induce Foxp3 expression. Furthermore, our

studies revealed that this depletion originates from the

induction of EAA consuming enzymes in APCs during

their recognition by regulatory T cells. The resulting

depletion of EAA then induces further Foxp3 during

antigen presentation to conventional T cells (Tconv),

converting them to Treg, which provides a mechanism

by which Foxp3+ Treg can mediate infectious tolerance

[24�]. Since EAA depletion is thought to specifically

inhibit TORC1 and not affect TORC2 activity, these

results seem to contradict the hypothesis that TORC2

inhibition is key to Foxp3 induction. These obvious

discrepancies might be explained by variations in the

quality and specificity of mTOR inhibition that is

caused by Rapamycin, EAA starvation and genetic

deletion. Furthermore the development of an ATP

competitive mTOR inhibitor (Torin1) has revealed

Rapamycin resistant TORC1 functions that might be

involved in regulating Foxp3 [25]. Therefore, the differ-

ential contribution of the TORCs to the induction of

Foxp3 is still unclear and downstream targets of the

TORC complexes involved in the induction have yet to

be identified. A possible mechanism that links mTOR


and Foxp3 expression was proposed by experiments

demonstrating the importance of the Foxo transcription

factors in the TGFb mediated induction of Foxp3 [26–28]. Harada and colleagues indicate that the PI3K-Akt-

mTOR axis might have a crucial role in controlling the

activity of the Foxo factors as inhibition of PI3K in T

cells lacking Cbl-b, a molecule known to reduce PI3K

activation, restored Foxp3 induction in these cells [26].

The activity of the Foxos is mainly controlled by their

translocation between nucleus and cytoplasm, which is

at least in part dependent on phosphorylation events

mediated by Akt [29]. Considering the accumulating

evidence that Akt and mTOR are important regulators

of Foxp3 expression and since PI3K inhibition inevita-

bly reduces Akt and mTOR activity, one can speculate

that these two downstream targets of PI3K modulate the

activity of Foxo factors to control Foxp3 expression.

Moreover, it will in due course be interesting to deter-

mine whether other conditions known to inhibit mTOR

such as glucose deprivation can also facilitate Foxp3

induction. Along these lines, Jurkat T cells seem to up-

regulate Foxp3 in a HIF-1a dependent manner when

cultured under hypoxic conditions known to activate

AMPK.

Further evidence for the importance of the mTOR path-

way in the induction and maintenance of Foxp3+ regu-

latory T cells in vivo comes from studies implicating

programmed death 1 protein (PD1). Ligation of PD1

during T cell activation in vitro was shown to inhibit

TORC1 and TORC2 and lead to an increase of Foxp3+

cells [30]. In addition to Foxp3 induction, several groups

have described that TORC1 inhibition by Rapamycin

favours Treg expansion over effector T cells (Teff) by

differentially regulating signalling and sensitivity to apop-

tosis [31–35]. It therefore seems likely that a combination

of inductive events and preferential expansion of Foxp3+

Treg contributes to the shift of T cell fate towards regu-

latory subsets.

T cell metabolism and anergyBesides the induction of regulatory T cells, T cell anergy

is thought to be another crucial tolerance inducing mech-

anism. The term anergy describes the secondary unre-

sponsiveness of T cells to a normally activating stimulus

via the TCR and CD28 with respect to cytokine pro-

duction and proliferation. This phenomenon was

ascribed to a lack of costimulation via CD28 during

the primary exposure to antigen resulting in suboptimal

activation of the Akt-mTOR pathway and incomplete

recruitment of transcription factors, particularly those

involved in IL-2 transcription. This leads to the induc-

tion of an anergic gene expression profile and failure of

the cell to enter the cell cycle [36]. Recently a number of

groups have extended this model by demonstrating that

signal 1 (TCR) and signal 2 (costimulation) are both

necessary to fully activate mTOR and to adjust the



cellular metabolism in order to meet the demands that

cell division and differentiation impose on the cell.

Zheng and colleagues further demonstrated that anergy

induction, by signal 1 alone, not only results in metabo-

lically anergic cells but that nutrient availability sensed

by the mTOR pathway controls anergy induction [37�].The authors convincingly show that anergy is associated

with a failure to up-regulate nutrient transporters such as

CD71 (transferrin receptor; iron uptake), CD98 (amino

acid uptake) and Glut1 (glucose uptake) and an inability

to induce glycolytic pathways. The blockade of leucine,

glucose and energy metabolism using N-acetyl-leucine

amide (NALA), 2-deoxyglucose (2DG) and 5-aminoimi-

dazole-4-carboxamide ribonucleoside (AICAR) were

able to induce anergy even in the presence of sufficient

signal 1 plus 2.

Besides these striking effects of mTOR inhibition that

seem to explain the potent immunosuppressive effects of

Rapamycin in vivo, recent reports indicate that the bac-

terial metabolite can also promote the development of

CD8+ memory T cells [38�,39��,40]. Administration of

Rapamycin in vivo enhances not only the magnitude, but

also the quality, of highly functional memory CD8+ cells.

Araki et al. were able to prove that this effect of Rapa-

mycin was due to TORC1 inhibition in CD8+ T cells (and

not other immune cells) by performing a series of knock-

down experiments targeting TORC1 components raptor

and FKBP12 as well as TORC1 downstream targets S6K1

and eIF4E in CD8+ cells [38�]. Similarly, Rao and col-

leagues demonstrate that inhibition of TORC1 reverses

the IL-12 induced skewing of CD8+ cell differentiation

into effector rather than memory type cells by disruption

of persistent T-bet expression. The reduced levels of the

transcription factor T-bet augment the induction of yet

another transcription factor, Eomesodermin, that has

been shown to favour CD8+ memory precursor cell

generation [39,41]. Additionally, the pharmacological

activation of the negative TORC1 regulator AMPK by

the anti-diabetic drug metformin, was able to reverse the

defects in lipid metabolism and memory cell develop-

ment seen in TRAF6 deficient T cells. This study

elegantly highlights the connection between lipid

metabolism, growth factor signalling and the mTOR

pathway [40]. Further evidence for the role of mTOR

in regulating CD8+ T cell differentiation comes from

studies investigating the role of the Wnt pathway [42].

Wnt signalling, which is known to activate TORC1

signalling by preventing GSK3 mediated TSC1/2 phos-

phorylation [43], was demonstrated to arrest effector T

cell differentiation at the expense of increased CD8+

memory stem cells [42]. The remarkable difference in

the response of CD8+ and CD4+ T cells to mTOR

inhibition suggests that mTOR signalling might play a

different role in controlling cellular fate in these two

subsets. Araki et al. speculate that this discrepancy could

be explained by the incomplete inhibition of mTOR


signalling by Rapamycin compared to genetic deletion

[44].

mTOR signalling and T cell traffickingWhile the majority of studies have focused on mTOR

inhibition and its role in the induction of Foxp3 in per-

ipheral T cells, Liu and colleagues identified lysopho-

spholipid sphingosine 1-phosphate (S1P) as novel

regulator of the Akt-mTOR axis in T cells [45]. The

S1P receptor is known to be an important regulator of T

cell trafficking and exit from the lymph nodes, and is the

inhibitory target of the novel immunosuppressive agent

FTY720 [46]. Interestingly, by activating mTOR S1P re-

ceptor signalling inhibits Foxp3 induction and suppressive

acitivity in thymocytes in a Rapamycin sensitive manner.

Further, Liu et al. demonstrated differential control of S1P

receptor expression upon stimulation of Tconv (abrupt

decrease) opposed to Treg (slower decrease). With the Treg

being released from S1P induced mTOR activity later,

Tconv can initiate the immune response relatively unim-

peded [45].

Sinclair et al. have investigated the effects of the PI3K-

mTOR axis on the expression of CD62L and CCR7, two

other important regulators of lymphocyte trafficking.

TORC1 inhibition by Rapamycin prevented IL-2

induced down-regulation of CD62L mRNA and CCR7

surface expression on activated CD8+ T cells in vitro, so

enabling these cells to retain their homing capacity for

secondary lymphoid organs in vivo [47�]. Similar to the

effects of FTY720, this sequestration of CD8+ effector T

cells away from sites of inflammation might also contrib-

ute to the immunosuppressive effects of Rapamycin invivo. Studies investigating the chemotactic response of

PBMC derived CD4+ cells treated with Rapamycin also

demonstrate that TORC1 signalling is necessary for

CCR5 mediated chemotaxis. In this case TORC1 activity

allows translation of a subset of mRNAs necessary for

chemotaxis (including cyclin D1 and MMP-9) through its

effect on the downstream target 4E-BP1 [48].

ConclusionsIn summary, T cells link instructive signals emerging

from the local microenvironment to their nutritional and

energetic status primarily by using a common route, via

mTOR, to control the functional fate of the cell. This

might represent a mechanism that allows lymphocytes

not only to differentiate according to their instructed fate

but also to fine-tune their response according to their

metabolic competence. The activation status of mTOR in

T cells has been demonstrated to crucially influence the

balance of inflammatory and tolerogenic T cell subset

differentiation. Understanding how the mTOR com-

plexes regulate T cell fate in greater detail opens up

the prospect of developing novel therapeutic regimens

(based on mTOR and other metabolic inhibitors) to treat

diseases in which this balance needs to be modified.



AcknowledgementsWe thank the Medical Research Council and the Edward Penley AbrahamTrust for their support.

References and recommended readingPapers of particular interest published within the period of review havebeen highlighted as:

� of special interest

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19.��

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This study expanded the widely accepted two-signal model of T cellactivation by emphasizing the importance of the up-regulation of themetabolic machinery by these signals. The experiments convincinglydemonstrate that anergic T cells are metabolically anergic and thatblockade of metabolic pathways can render T cells anergic.

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The authors demonstrate that administration of Rapamycin in LCMVinfected mice exerts immunostimulatory effects by enhancing the quan-tity and quality of CD8+ memory T cells. Furthermore, they establish that


this is a T cell intrinsic effect by demonstrating that knock down of mTORpathway components can induce similar differentiation patterns.

39.��

Rao RR, Li Q, Odunsi K, Shrikant PA: The mTOR kinasedetermines effector versus memory CD8+ T cell fate byregulating the expression of transcription factors T-bet andEomesodermin. Immunity 2010, 32:67-78.

Rao et al. provide evidence that TORC1 inhibition by Rapamycin pro-motes memory CD8+ T cell differentiation by blocking IL-12 inducedsustained T-bet expression and promoting Eomesodermin expression.

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The studies performed by Sinclair et al. revealed that inhibition of thePI3K-mTOR axis can prevent activation and cytokine induced down-regulation of CD62L and CCR7 surface expression.

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mtor signalling and metabolic regulation of t cell differentiation

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