How does the mammalian target of rapamycin (mTOR) influence CD8 ...

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Cell Cycle 9:15, 2952-2957; August 1, 2010; © 2010 Landes Bioscience

How does the mammalian target of rapamycin (mTOR) influence CD8 T-cell differentiation? Robert J. Salmond and Rose Zamoyska* Institute for Immunology and Infection Research; The University of Edinburgh; Edinburgh, UK

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aive T lymphocytes maintain a quiescent resting state until they encounter antigen whereupon they undergo a switch in their metabolic program in preparation for proliferation and differentiation. This activation process involves a dramatic upregulation of protein synthesis that is essential for cell growth and the differentiation of effector function. An essential regulator of protein synthesis in T cells is the mammalian target of rapamycin (mTOR), a serine/threonine kinase that regulates both the availability of amino acids and the process of cap-dependent translation. Recent data indicate that mTOR influences activation and cell fate determination in T cells. We discuss these findings in light of what is currently known about the function of mTOR and its targets in CD8 T cells. Introduction

Key words: rapamycin, mTOR, CD8, T cells, T-cell signaling, T-cell differentiation Submitted: 05/11/10 Accepted: 05/12/10 Previously published online: www.landesbioscience.com/journals/cc/ article/12358 DOI: 10.4161/cc.9.15.12358 *Correspondence to: Rose Zamoyska; Email: [email protected]

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Naïve T cells encounter antigen in secondary lymphoid organs and recognition via the T-cell receptor (TCR) of cognate peptide-MHC complexes on the surface of antigen-presenting cells (APCs) can result in a variety of responses. Signals transduced through the TCR are integrated with signals from co-stimulatory molecules, cytokine and chemokine receptors and together direct the outcome of the response. CD4 T cells can differentiate to a number of effector fates that can be delineated by distinct cytokine secretion profiles, whereas CD8 T cells gain cytotoxic capacity and the ability to destroy virally infected cells. Both CD4 and CD8 T cells can also develop into ­long-lived memory

cells that are critical for a rapid response upon secondary encounter with an infectious agent. For both CD4 and CD8 T cells, the processes of effector and memory cell differentiation are influenced by the nature of the antigen and the cytokine milieu. Increasing evidence has placed the serine/threonine (Ser/Thr) kinase mammalian target of rapamycin (mTOR) as a critical regulator of T-cell proliferation and determinant of effector versus memory cell fate. The role of mTOR in CD4 T-cell function has been reviewed in a number of recent articles,1-3 therefore the focus of the present paper is on the emerging role of mTOR as a critical regulator of signal integration in CD8 T cells. Furthermore, we discuss the importance of specific pathways activated downstream of mTOR in T cells. Two Intracellular mTOR Complexes Differ in Sensitivity to Rapamycin mTOR is the catalytic component of at least two distinct protein complexes (Fig. 1). The first, mTOR complex 1 (mTORC1) was originally identified as being the target of the macrolide immunosuppressant rapamycin. mTORC1 is composed of mTOR, regulatory associated protein of mTOR (raptor) and mammalian lethal with sec-13 protein 8 (mLST8).4-6 A raptor-associated protein proline-rich Akt substrate of 40 kDa (PRAS40) negatively regulates mTORC1 activity7 whilst a recently described 48 kDa protein DEP domain-containing protein associated with mTOR (DEPTOR) interacts directly with mTOR and inhibits

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Figure 1. mTOR signaling pathways in T cells. Stimulation of T cells through TCR triggering in combination with signals transduced by costimulatory CD28 molecules and cytokine receptors results in activation of PI3K. PI3K activity facilitates recruitment of phosphoinositide-dependent kinase (PDK) 1 and Akt to the plasma membrane. PDK1-dependent phosphorylation of the active loop Thr308 residue results in activation of Akt. Akt phosphorylates TSC2, preventing formation of TSC1/TSC2 heterodimers, which in turn relieves inhibition of the small GTPase Rheb. Rheb antagonizes the endogenous mTOR inhibitor FKBP38 allowing activation of mTORC1. In addition, TSC1/TSC2 facilitates activation of mTORC2 independently of effects on Rheb activity. mTORC1 activity results in phosphorylation of downstream effectors 4E-BP1 and S6K1; S6K1 might regulate mTORC2 through phosphorylation of Rictor. mTORC2 is required for maintenance of phosphorylation and expression of PKCa and also promotes Akt activity through phosphorylation of residue Ser473. PRAS40 binds Raptor and negatively regulates mTORC1 whilst Deptor inhibits the activity of both mTORC1 and mTORC2.

activation of both mTOR complexes.8 The best-characterized mTORC1 substrates are the translational inhibitory protein 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In T cells, mTORC1 is activated downstream of the TCR, CD28, cytokine receptors and chemokine receptors in a phosphoinositide 3-kinase (PI3K)-dependent manner. Although the precise mechanism by which mTORC1 is activated in T cells has not been assessed directly, it is thought that the PI3K-dependent

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Ser/Thr kinase Akt mediates mTORC1 activation through phosphorylation of tuberous sclerosis complex 2 (TSC2),9 which prevents TSC2 heterodimerization with TSC1. TSC1/2 heterodimers demonstrate GTPase activating protein (GAP) activity towards the small GTPase Rheb.10 Akt-mediated inhibition of TSC1/2 heterodimer formation enables GTPbound Rheb to activate mTOR through antagonizing the endogenous mTOR inhibitor FK506 binding protein 38 (Fig. 1).11

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mTOR complex 2 (mTORC2) also contains mLST8 but in place of raptor is the rapamycin insensitive companion of mTOR (rictor) and mammalian stressactivated protein kinase interacting protein 1 (SIN1).12-14 mTORC2 is required for the phosphorylation and maintenance of expression of protein kinase Cα.14,15 In addition, mTORC2 influences activation of Akt by phosphorylating Akt on Ser473.15,16 The mechanism by which mTORC2 itself is activated is unclear but may require the activity of the TSC1/2 complex, independent of its effects on Rheb.17 Therefore the TSC1/2 complex appears to negatively regulate mTORC1 but positively regulate mTORC2. Furthermore, there is evidence that the two canonical mTOR complexes might regulate each other. For example, mTORC2 might indirectly affect the activity of mTORC1 through phosphorylation and activation of Akt whilst the mTORC1 substrate S6K1 has been shown to phosphorylate rictor which may in turn affect mTORC2 activity18 (Fig. 1). Intriguing data from the Croft laboratory has shown that, in T cells, mTOR can also interact with an additional Ser/ Thr kinase aurora B.19 These authors suggest that aurora B is important for phosphorylation of 4E-BP1 and S6K1.19 It is not clear whether aurora B can directly phosphorylate 4E-BP1 or S6K1 or whether it regulates the activity of mTOR towards these substrates. Similarly to the classical mTORC1, the mTOR-aurora B complex is inhibited by rapamycin. Further evidence for the existence of such a non-canonical mTOR complex has come from recent experiments in which the effects on translation of knockdown of mTOR, rictor and raptor expression were compared.20 Knockdown of mTOR expression in adipocytes resulted in translational repression of mRNAs containing a 5' oligopyrimidine tracts whereas knockdown of raptor or rictor had only a small effect on translation of these mRNAs.20 These data suggest that in some circumstances mTOR might exert its effects independently of formation of the classical mTORC1 and mTORC2 complexes. A wealth of data has accumulated demonstrating the importance of mTOR in the immune system through studying

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the effects of rapamycin. Rapamycin associates with the cytosolic FK506 binding protein of 12 kDa (FKBP12) and the rapamycin-FKBP12 complex binds to and inhibits the activity of mTORC1.21‑23 Rapamycin-FKBP12 does not directly bind mTORC2 but prolonged treatment with rapamycin may prevent formation of mTORC2.24 Rapamycin is currently licensed for immunosuppressive therapy in humans following transplantation and is in trials testing its efficacy as an anticancer therapy. Rapamycin treatment induces CD4 T-cell anergy25,26 whilst genetic ablation of mTOR (encoded by FRAP1) expression results in a failure of CD4 T cells to proliferate in response to TCR triggering or to differentiate to any of the T helper lineages.27 Furthermore, rapamycin has immunomodulatory effects on B cells, NK cells, dendritic cells, mast cells and neutrophils (reviewed in ref. 2 and 3). By contrast, data has suggested that, in some circumstances, CD8 T-cell proliferation is resistant to the inhibitory effects of rapamycin28,29 whilst intriguing data have indicated a critical role for mTOR in determining whether CD8 T cells adopt an effector versus memory cell fate.30,31 Roles of mTOR Signaling in CD8 T Cells Naïve CD8 T cells encounter antigen in lymph nodes draining the site of an infection. Dendritic cells ‘licensed’ through CD4 T helper cell provision of CD40L-CD40 interactions,32 or activated by inflammatory signals drive the activation and differentiation of naïve CD8 T cells through presentation of cognate antigen and the production of important cytokines such as interleukin (IL)-12. The availability of additional cytokines including IL-2 and IL-15 is also critical in determining the outcome of CD8 T-cell activation.33 mTOR is activated both through the TCR and following cytokine receptor triggering and has been shown to be important for the response of CD8 T cells to these stimuli. Upon encounter of naïve T cells with cognate peptide presented by activated APCs, signals transduced through the TCR and CD28 co-receptor induce T-cell

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growth and cell cycle entry. Inhibition of TCR/CD28-induced mTOR activation by rapamycin reduces cell growth and delays entry of CD4 T cells into the cell cycle.1,34 Interestingly, Slavik and colleagues have shown that some human CD8 T-cell clones are resistant to the anti-proliferative effects of rapamycin, even when the drug is used at very high (micromolar) concentrations.28 In these experiments, rapamycin-resistant CD8 T-cell proliferation was associated with increased expression of the anti-apoptotic Bcl-2 family member Bcl-X L and was completely blocked through inhibition of PI3K.29 These data suggest that mTOR is dispensable for TCR-driven CD8 T-cell proliferation. However, the recent development of potent “active site” mTOR inhibitors has highlighted the existence of rapamycin-resistant mTOR activities.35,36 Murine CD4 T cells genetically deficient in mTOR expression display markedly defective TCR-induced proliferation;27 we await similar experiments to be performed on purified CD8 T cells to provide categorical determination of the role of mTOR in T-cell proliferation. Following initial antigenic stimulation, clonal expansion of CD8 T cells is driven by mitogenic cytokines including IL-2 and IL-15. This process facilitates a rapid and dramatic increase in the number of antigen-specific T cells available to participate in an immune response. However, additional signals are required for CD8 T cells to gain effector function. In this regard, expression of the transcription factor T-bet is critical for CD8 T cells to gain cytolytic activity and express effector cytokines such as interferon-γ (IFNγ) (reviewed in ref. 37). TCR signals and IL-12 produced by DCs and macrophages are important in elevating and sustaining the level of T-bet expression in CD8 T cells. A recent study by Rao et al. showed that IL-12 enhanced and sustained TCRinduced mTOR activation in OT-1 TCR transgenic CD8 T cells whilst rapamycin treatment reversed IL-12-induced T-bet expression and reduced target cell lysis and IFNγ production.31 Interestingly the authors also reported that concomitant to suppression of T-bet expression, rapamycin treatment resulted in sustained and elevated expression of the transcription

factor eomesodermin.31 Importantly the balance of expression of eomesodermin and T-bet has been shown to be critical in determining whether CD8 T cells adopt memory or effector cell fates; high T-bet expression favours effector differentiation whilst high levels of eomesodermin are proposed to induce memory cell differentiation.38 In addition to gaining cytolytic activity and the capability to secrete cytokines such as IFNγ, effector CD8 T cells gain the ability to migrate from the lymph nodes to the site of infection. Expression of CD62 ligand (CD62L; L-selectin) and the chemokine receptor CCR7 are important for the homing of naïve and selfrenewing, recirculating, central memory T cells to lymph nodes and is lost in effector cell populations. A wealth of data implicates IL-2- and IL-15-dependent signals in the regulation of this aspect of effector/ memory CD8 T-cell differentiation. For example, several groups have reported that culture of TCR transgenic CD8 T cells in the presence of high doses of IL-2 favored the generation of effector cells, characterized by elevated cell surface expression of the activation marker CD44 and low expression levels of CD62L and CCR7,39,40 over memory cells. By contrast, low doses of IL-2,40 or culture in the presence of IL-15,39 resulted in differentiation of CD8 T cells to a memory-like phenotype (CD44 high CD62Lhigh CCR7 high). In addition, experiments by the Ahmed group have shown that the duration of IL-2 signaling during differentiation also affects the generation of anti-viral CD8 T-cell effector and memory responses in vivo.41 Rapamycin inhibits IL-2dependent downregulation of CD62L and CCR7 expression in vitro, indicating the importance of IL-2-induced mTOR activation in this process.42 Interestingly, whilst both IL-2 and IL-15 trigger mTOR activation, IL-2-induced mTOR activity is relatively sustained.43 Thus, IL-2 but not IL-15 signaling induces prolonged mTOR activation that in turn results in downregulation of CD62L and CCR7 expression and biases differentiation to an effector rather than memory fate. Rapamycin has long been regarded as a potent immunosuppressant. However, recent intriguing data have shown that

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the effects of rapamycin are highly dosedependent and that, in some circumstances, the drug might act to enhance anti-viral responses in vivo. Ahmed and co-workers recently reported that treatment of mice with high doses of rapamycin (600 µg/kg/day) inhibited CD8 T-cell responses during infection with lymphocytic choriomeningitis virus (LCMV), as expected.30 Surprisingly, lower doses of rapamycin had stimulatory effects on memory CD8 T-cell generation. Virusspecific memory CD8 T-cell numbers were increased in rapamycin-treated mice. Moreover, memory cells generated in the presence of rapamycin were also qualitatively distinct, expressing higher levels of CD62L, CD127 and anti-apoptotic Bcl-2, lower levels of KLRG1, and were superior in their ability to undergo homeostatic proliferation and to make recall responses upon a second encounter with virus.30 Further experiments showed that antigen-specific CD8 T cells in which raptor expression had been knocked down using RNA interference displayed a similar cell surface phenotype to that seen following rapamycin treatment, indicating that mTORC1 was important in the regulation of memory cell differentiation.30 Taken together, these data indicate that the level of mTOR activation is critical in determining the outcome of CD8 T-cell activation both in vitro and in vivo. Strong activation of mTOR through TCR triggering in combination with cytokines such as IL-2 and IL-12 potently induces T-bet expression, the gain of cytolytic activity, effector cytokine secretion and the downregulation of lymph node homing receptors. By contrast, a lower level of mTOR activation as occurs during IL-15 stimulation or in the presence of low levels of rapamycin favours memory cell differentiation. Thus mTOR activity acts as a critical rheostat determining naïve CD8 T-cell fate. Molecular Mechanisms of mTOR Signaling in T Cells The immunosuppressive activity of rapamycin has been recognized for two decades, yet the molecular mechanisms by which rapamycin and mTOR regulate T-cell function are relatively poorly

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understood. One mechanism by which mTOR controls T-cell growth is through regulation of protein synthesis. For example, IL-2 stimulation of T cells potently activates mTOR and stimulates uptake of amino acids, protein synthesis and subsequently enables maintenance of a large cell size.39,43 By contrast, rapamycin decreases T-cell blast size. Microarray analysis of polysome-associated and total cellular mRNA indicated that rapamycin reduced translation of the majority of expressed genes in Jurkat T cells whilst translation of a small subset of genes was completely inhibited by rapamycin.44 The mechanism by which mTORC1/rapamycin control this process, is likely to involve regulation of the phosphorylation of 4E-BP1. 4E-BPs negatively regulate protein synthesis by binding to and sequestering the mRNA cap-binding protein eukaryotic initiation factor 4E (eIF4E).45 Phosphorylation of 4E-BP1 by mTORC1 prevents its association with eIF4E and enables translation initiation.46,47 A recent study indicated that oncogenic mTOR signals that were obligatory for T-cell lymphomagenesis were primarily mediated through the 4E-BP-eIF4E axis,48 consistent with a critical role for mTOR/rapamycin mediated translational regulation. Whilst the role of 4E-BP phosphorylation in mTOR signaling in non-transformed T cells has yet to be directly assessed, regulation of gene expression at the level of translation has been suggested to be of particular importance in T cells.49 Furthermore, recent data from the Sonenberg group have shown the vital importance of translation regulation mediated via the 4E-BPs in the innate immune response to viral infection.50 A further mechanism by which mTOR might regulate translation in T cells is through the S6K1-dependent phosphorylation of ribosomal protein (rp) S6. We have shown that rpS6 phosphorylation acts as a point of convergence for the mTOR/S6K1 and extracellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathways in CD8 T cells following TCRtriggering.51 Interestingly, phosphorylation of specific serine residues with rpS6 was regulated differentially by the MAPK and mTOR pathways. In this regard,

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TCR-induced phosphorylation of rpS6 residues Ser240/244 was predominantly mediated by mTOR-dependent pathways, whereas phosphorylation of residues Ser235/236 was mediated downstream of both MAPK and mTOR.51 Although the role of phosphorylation in rpS6 function is not clear, expression of rpS6 is critical for ribosome biogenesis and homozygous deletion of the rpS6 gene in immature CD4 + CD8 + thymocytes completely blocks T-cell development.52 Analysis of ‘knockin’ mice expressing a nonphosphorylatable rpS6 protein53 indicates that mTOR and ERK-dependent rpS6 phosphorylation is dispensable for T-cell development (Salmond, Meyuhas, Zamoyska, unpublished data). Nonetheless, Hsieh et al. have recently reported that global protein synthesis in thymocytes from rpS6 knockin mice is reduced by 20% relative to wild-type levels,48 indicating a role for rpS6 phosphorylation in this process. Additional studies using the rpS6 knockin mouse system will be required to determine the impact of mTOR-mediated rpS6 phosphorylation in T-cell differentiation and effector function. It is clear that rapamycin and mTOR also regulate gene expression at the level of transcription. Cytokine receptors stimulate gene expression though activation of Jak kinases and subsequent Jak-dependent phosphorylation of signal transducer and activator of transcription (STAT) transcription factors.54 Several reports have shown that mTOR and rapamycin can regulate cytokine-induced STAT activation. In this regard, Kusaba and colleagues reported that IL-12-induced STAT3 phosphorylation was reduced by rapamycin treatment.55 Similarly Delgoffe et al. showed that IL-12induced STAT4, IL-6-induced STAT3 and IL-4-induced STAT6 phosphorylation was diminished in mTOR-deficient T cells.27 In the absence of mTOR, IL-12-induced, STAT4-dependent T-bet and IL-4-induced, STAT6-dependent GATA-3 mRNA expression were severely impaired.27 It has been suggested that mTOR might regulate STAT phosphorylation through a direct interaction that is disrupted by rapamycin55 or in some circumstances might directly phosphorylate STAT molecules.56

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Concluding Remarks The importance of the mTOR signaling pathway in the immune system has been recognized for many years. Research from the past decade has highlighted the complexity of mTOR signaling through the discovery of multiple mTOR complexes whose composition, regulation and downstream effectors are distinct. Furthermore, recent data have shown that rapamycin, primarily regarded as an immunosuppressive drug, can have stimulatory effects on certain aspects of CD8 T-cell function. However, much remains to be discovered before we can fully understand the role of mTOR function in T cells. The biochemical mechanisms by which mTOR and rapamycin control T-cell activation and differentiation are poorly understood. Analysis of the roles of downstream effectors of mTOR, such as 4E-BPs, S6K1 and rpS6 phosphorylation, using mice genetically deficient in these proteins will prove informative. Furthermore, the specific roles of mTORC1 and mTORC2 in T cells can now be addressed using newly available conditional knockout mice.27 A fuller understanding of the mechanisms of mTOR function may suggest more specific and subtle means of clinical intervention for the modulation of immune responses in humans.57 References 1. Mondino A, Mueller DL. mTOR at the crossroads of T-cell proliferation and tolerance. Semin Immunol 2007; 19:4-6. 2. Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol 2009; 9:324-37. 3. Weichhart T, Saemann MD. The multiple facets of mTOR in immunity. Trends Immunol 2009; 30:218-26. 4. Kim DH, Sarbassov DD, Ali SM, Latek RR, Guntur KV, Erdjument-Bromage H, et al. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell 2003; 11:895-904. 5. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002; 110:163-75. 6. Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002; 110:177-89. 7. Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E, et al. PRAS40 is an insulinregulated inhibitor of the mTORC1 protein kinase. Mol Cell 2007; 25:903-15.

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