p110δ Couples the IL-2 Receptor to Akt but IL-2-Mediated T Cell Proliferation Is p110δ Independent

TCR-primed CD8⁺ T cells cultured in IL-2 clonally expand and differentiate to effector cytotoxic T cells (CTL) (Manjunath et al., 2001). This model mimics the in vivo situation where sustained IL-2 signaling promotes the production of terminally differentiated effector cytotoxic T cells (Kalia et al., 2010; Malek and Castro, 2010; Pipkin et al., 2010). IL-2 strongly activates Akt and antigen-experienced T cells cultured in IL-2 contain high amounts of Akt phosphorylated on the 3-phosphoinositide-dependent kinase 1 (PDK1) substrate site T308 (Figure 1A). The TCR activates Akt via a PI3K complex that contains the p110δ catalytic subunit (Garçon et al., 2008).Figure 1A shows that p110δ PI3K also coupled the IL-2 receptor to Akt. Hence, T cells with wild-type p110δ substituted with a catalytically inactive mutant, p110δ^D910A, do not phosphorylate Akt T308 in response to IL-2 and thus lack Akt activity as judged by the loss in phosphorylation of Foxo transcription factors (Figure 1A).

The sustained proliferation and survival of antigen-primed CTL is dependent on exogenous IL-2 (Figure 1B). However, activated p110δ^D910A-expressing T cells proliferated normally to IL-2 (Figure 1C). Further experiments examined the impact of a p110δ inhibitor, IC87114, on IL-2-induced Akt activity in antigen-activated P14 TCR transgenic T cells. Triggering of the TCR expressed on these cells with a peptide from the lymphocytic choriomeningitis virus (LCMV), glycoprotein gp33-41, presented by the MHC class I molecule H-2D^b, followed by clonal expansion in IL-2, generated a homogenous population of CTL. The exposure of IL-2-maintained P14 CTL to IC87114 caused loss of Akt T308 phosphorylation (Figure 1D) but did not prevent T cell proliferation (Figure 1E).

To explore further the role of Akt in IL-2 proliferative responses, a set of experiments with AktI, a selective allosteric inhibitor of all three isoforms of Akt, were performed. AktI prevents the essential PDK1-mediated phosphorylation of Akt on T308 (Green et al., 2008). The addition of AktI to CTL caused loss of Akt T308 phosphorylation and a resultant loss of Akt activity as judged by the loss in phosphorylation of the Akt substrates Foxo1 and Foxo3a (Figure 1F). However, AktI did not prevent IL-2-induced proliferation of the CTL nor did it cause cell death (Figure 1G). Hence, CTL do not need active Akt to proliferate and survive.

PDK1 Is Required for T Cell Proliferation

One explanation for the Akt independence of IL-2-induced proliferation is that other members of the AGC family of kinases might compensate for loss of Akt function. We therefore examined the impact of deleting the gene encoding PDK1,Pdpk1, on IL-2 signal transduction. PDK1 phosphorylates and activates multiple AGC kinases such as RSK and S6K1 in a response that is independent of PI(3,4,5)P₃ production (Bayascas, 2008).Pdpk1 deletion is thus a strategy that simultaneously inactivates Akt- and PI(3,4,5)P₃-independent AGC kinases in T cells (Kelly et al., 2007). Accordingly, mice expressing floxedPdpk1 alleles (Pdpk1^fl/fl) were backcrossed with C57BL/6-GT(ROSA)26^{tm9 (creEsr1) Arte} mice (TamoxCre) that express a tamoxifen-regulated Cre recombinase in the ROSA locus.Pdpk1^fl/fl TamoxCre CTL were then generated and treated with 4-hydroxytamoxifen (4OHT) to delete the floxedPdpk1 alleles.Pdpk1 deletion in 4OHT-treatedPdpk1^fl/fl TamoxCre CTL was confirmed (Figure 2A). Importantly, immunoblot analysis also revealed that 4OHT-treatedPdpk1^fl/fl TamoxCre CTL had lost phosphorylation of RSK on the PDK1 target site S227 and had lost phosphorylation of Akt on the PDK1 target site T308. The loss of Akt and S6K1 catalytic activity was judged by the loss of phosphorylated Foxos and the ribosomal S6 subunit (Figure 2B).

Pdpk1 deletion in CTL thus eliminated the activity of multiple AGC kinases. We therefore assessed the ability of IL-2 to sustain survival and proliferation ofPdpk1-null cells.Figure 2C shows thatPdpk1-null CTL failed to proliferate in response to IL-2. However, whereas IL-2 deprivation resulted in rapid cell death of CTL (Figure 1B), the loss of PDK1 did not cause cell death (Figure 2D). PDK1 is thus essential for IL-2-induced T cell proliferation but not for cell survival.

IL-2 and PDK1 but Not Akt Sustain Glucose Uptake in CTL

In many cells Akt controls cell proliferation because it maintains glucose metabolism. In this context, as naive CD8⁺ T cells respond to cognate antigen and differentiate to CTL, they increase glucose uptake and become highly glycolytic. Glucose uptake in CTL was not autonomous but depended on the provision of exogenous IL-2 (Figure 3A). Moreover, the importance of glucose uptake for T cell proliferation was easily demonstrated: lowering of exogenous glucose concentrations severely impaired IL-2-induced proliferative responses (Figure 3B). That glucose deprivation but not Akt inhibition prevented IL-2-induced CTL proliferation argues that Akt activity is not required for IL-2-driven glucose uptake.Figure 3C addresses this point and shows that loss of Akt activity had no impact on the ability of IL-2-cultured CTL to take up glucose. The Akt independence of IL-2-induced T cell proliferation can thus be explained by the ability of IL-2 to maintain glucose uptake via Akt-independent pathways. We then assessed whether PDK1 plays a role in IL-2-induced glucose uptake.Figure 3D shows that PDK1-null T cells had a strikingly reduced rate of glucose uptake but no defects in amino acid uptake (Figure 3E). Hence PDK1, but not Akt, is essential for IL-2 control of glucose uptake.

The Akt independence of IL-2-induced glucose uptake raised the question whether Akt activity was required for the initial increase in glucose metabolism initiated by the TCR in CD8⁺ T cells.Figure 3F shows that TCR triggering stimulated a large increased glucose uptake and this was unimpaired in cells treated with the Akt inhibitor AktI or the PI3K p110δ inhibitor IC87114. The regulation of glucose uptake via the T cell antigen receptor in CD8 T cells is thus not mediated by Akt.

PDK1 and Akt Transcriptional Programs in CTL

If Akt is not essential for glucose uptake or IL-2-mediated T cell survival or proliferation, what does it do? This question is important because sustained IL-2 signaling is required to maintain CTL effector function both in vitro and in vivo (Kalia et al., 2010; Pipkin et al., 2010) and there is clearly the potential for Akt to mediate the effects of IL-2 on CTL function. Accordingly, we used Affymetrix microarray analysis to transcriptionally profilePdpk1-null and Akt-inhibited CTL. Approximately 9400 annotated genes were expressed in CTL, and the impact of PDK1 loss or Akt inhibition was revealed by a decrease in the expression of less than 2%–3% of these transcripts and an increase in the expression of another 3%–4% (Tables S1 and S2 available online).

We first looked for non-Akt-dependent, PDK1-regulated genes to understand why PDK1 but not Akt is required for IL-2-induced T cell proliferation. Selected genes controlled only by PDK1 but not Akt are highlighted inFigure 4A (Table S3). Of particular note, PDK1 controlled expression of hexokinase 2, and hexokinases play a key role in effective uptake of exogenous glucose. We also used the NIAID DAVID website (http://david.abcc.ncifcrf.gov) to mine the gene ontology terms (GO) associated with the PDK1-controlled genes. We noted that GO terms related to lipid and cholesterol metabolism (Figure 4B) were strongly overrepresented in this list, indicating that PDK1 but not Akt controls lipid metabolism in CTL.

We next interrogated the data to characterize genes coregulated by PDK1 and Akt. One clear result was that Akt inhibition orPdpk1 deletion resulted in the re-expression of Foxo target genes (Figure 4C), including the IL-7 receptor α subunit (CD127), the transcription factor Klf2, and its targets L-selectin (CD62L), CC motif chemokine receptor 7 (CCR7), and sphingosine-1-phosphate receptor 1 (S1P1) (Carlson et al., 2006; Fabre et al., 2008; Kerdiles et al., 2009; Ouyang et al., 2009).Figures 4D and 4E used quantitative polymerase chain reaction (PCR) analysis to verify these changes. IL-2 activation of Akt is dependent on PI3K p110δ (Figure 1), so it would be predicted that inactivating p110δ would also restore expression of the Foxo-regulated genes in CTL. Indeed, IL-2-cultured CTL expressing catalytically inactive p110δ (Pik3cd^D910A) maintained high expression of Klf2 and CD62L, CCR7, and S1P1 (Figure 4F). Moreover, treatment of CTL with the p110δ inhibitor IC87114 drove re-expression of Klf2 and its targets (Figure 4G).

Inhibition of PI3K p110δ and Akt Reprograms CTL Trafficking

Deletion ofPdpk1 or the inhibition of Akt caused CTL to re-express CD62L and CCR7 (Figures 4C–4E). CD62L controls T cell adhesion to the endothelium of high endothelial venules and CCR7 directs migration of T cells into lymphoid organs. Moreover, the loss of CD62L and CCR7 by CTL is part of the program that directs the trafficking of CTL away from lymphoid tissue. The re-expression of CD62L and CCR7 in CTL deleted ofPdpk1 or exposed to AktI could argue that inhibition of Akt might reprogram the trafficking of CTL and restore their ability to transmigrate from the blood into secondary lymphoid tissue. In this context, strong activation of PI3K and Akt is required and sufficient to downregulate the ability of naive T cells to home to secondary lymphoid tissue in vivo (Finlay et al., 2009; Waugh et al., 2009). It is also known that p110δ activity is required for T cells to exit lymphoid tissue and home to sites of infection (Liu and Uzonna, 2010). T cells thus appear to need to activate Akt to exit lymphoid tissues. However, it is not known whether inhibition of Akt is sufficient to reprogram the ability of CTL to home to lymphoid tissue. In this context, inhibition of Akt had pleiotropic effects on the expression of multiple adhesion molecules and chemokine receptors by CTL and was not limited to controlling CD62L and CCR7 expression (Figure 5A).

To directly test the impact of Akt inhibition on CTL trafficking, in vivo adoptive transfer experiments were performed comparing the ability of wild-type CTL or CTL pretreated with either AktI or the p110δ inhibitor IC87114 to home from the blood to lymphoid tissue. Strikingly, CTL pretreated with either AktI or IC87114 showed strong preferential homing to lymph nodes and spleen compared to wild-type CTL (Figures 5B and 5C). Sustained activation of Akt is thus required to maintain the trafficking characteristics of CTL: Loss of p110δ catalytic activity or Akt inhibition reprograms the ability of CTL to traffic to lymphoid tissue.

To further explore the in vivo role of Akt in CD8 T cell trafficking responses, we compared in vivo immune responses of wild-type andPdpk1^K465E/K465E T cells. The K465E mutation creates a PDK1 molecule with a mutated PH domain that cannot bind PI(3,4,5)P₃ and can support only a low amount of Akt activity (Waugh et al., 2009).Figure 5D shows that both wild-type and K465E T cells proliferated in vivo in response to immunization with cognate antigen and lipopolysaccharide (LPS). However, K465E T cells failed to downregulate CD62L (Figure 5E) and were retained in lymphoid tissue (Figure 5F) compared to control effector CD8⁺ T cells.

Akt Is Necessary to Induce and Sustain Expression of CTL Effector Molecules

A key role for IL-2 in CD8⁺ T cells is to promote effector differentiation of CTL by driving expression of cytolytic effector molecules and by controlling the cytokine receptor profile of CTL (Janas et al., 2005; Pipkin et al., 2010). It was therefore striking that inhibition of Akt caused a decrease in the expression of mRNA encoding several molecules critical for CTL effector function (Figure 6A), notably multiple granzymes, Fas ligand, the cytolytic effector perforin, and interferon gamma (IFN-γ). It was equally notable that Akt inhibition had a considerable impact on the transcription of key cytokine receptors. Akt inhibition thus decreased expression of mRNA encoding IL-12 receptor β-chains while simultaneously increasing expression of mRNA encoding IL-6 receptors and CD27 (TNFRSF7), the CD70 coreceptor (Figure 6B). The role of Akt in perforin and IFN-γ expression was verified by real-time PCR analysis, ELISA, and immunoblot analysis (Figure 6C). Akt inhibition thus caused loss of IFN-γ mRNA expression and prevented IFN-γ protein production. Similar results were obtained after the deletion ofPdpk1 from CTL (Figure 6D), providing genetic evidence that PDK1-Akt signaling sustains perforin and IFN-γ protein expression in CTL.

Akt is also rapidly activated in CD8⁺ T cells in response to triggering of the TCR. Akt is not required for TCR-induced metabolic programs (Figure 3F), but is it necessary for the TCR to initiate expression of CTL effector molecules? In these experiments we focused on the induction of IFN-γ because this cytokine can be induced rapidly in response to triggering of the TCR in both naive and effector CD8⁺ T cells. Moreover, it has been shown that p110δ is required for TCR-induced IFN-γ production during immune activation but the role of Akt has not been addressed (Liu and Uzonna, 2010; Soond et al., 2010). We found that inhibition of Akt suppressed TCR induction of IFN-γ mRNA and protein in both naive CD8⁺ T cells and effector CTL (Figures 7A and 7B). This reflects that Akt activity is important for IFN-γ gene transcription.Figure 7C thus shows that the loss of Akt activity reduced the amount of RNA polymerase II (Pol II) recruitment to the IFN-γ transcription start site and distal exons.

Why is Akt needed for IFN-γ production? We considered the possibility that Akt controlled the activity of mTOR (mammalian target of rapamycin), which is known to control the expression of T effector molecules (Rao et al., 2010). However, althoughPdpk1 deletion prevented phosphorylation of the mTOR target S6 (Figure 2B), inhibition of Akt only weakly suppressed the phosphorylation of S6 and S6K1 (Figure S1). Akt activity is thus dispensable for mTOR activation in CTL. Accordingly, an inability to activate mTOR does not explain why Akt is required for IFN-γ production. However, an important insight as to a possible mechanism for Akt control of IFN-γ production came from experiments withPdpk1^K465E/K465E T cells that support only a low level of Akt activity. These T cells have a selective defect in the phosphorylation and inactivation of Foxo family transcription factors and fail to downregulate expression of Foxo gene targets (Waugh et al., 2009). Foxos can both transactivate and repress gene expression (Fabre et al., 2008), and in this context, TCR-induced production of IFN-γ is severely attenuated inPdpk1^K465E/K465E T cells (Figure 7D). This is consistent with a model whereby the defects in IFN-γ production caused by inhibition of Akt activity in CTL might be caused by relocation of Foxo transcription factors in the nuclei of T cells that then repress the IFN-γ gene. To assess whether the restoration of Foxos to the nuclei of TCR-activated T cells would impact on IFN-γ gene expression, we examined IFN-γ production in T cells expressing a GFP-tagged Foxo3a mutant with alanine substitutions at its Akt substrate sequences, T32, S252, and S314 (FoxoAAA). This phospho mutant could restore Foxo transcriptional function in cells expressing active Akt.Figures 7E and 7F show that activated T cells expressing the FoxoAAA mutant could not produce IFN-γ in response to peptide triggering of TCR complexes. Akt-regulated nuclear export of Foxo transcription factors is thus required for IFN-γ production.

Mice

C57BL/6GT(ROSA)26^{tm9(creEsr1)Arte} (TamoxCre) mice were obtained from Taconic Artemis Pharmaceuticals. Mice carrying floxedPdpk1 allelesPdpk1^flΔneo (PDK1^fl); OT1 and P14 TCR transgenic mice; P14 TCR transgenic mice carrying a knockin mutation for a substitution of lysine for glutamic acid at residue 465 in the PH domain of PDK1 (Pdpk1^K465E) andPik3cd^D910A mice containing a knockin mutation of PI3K wherein wild-type alleles of the p110δ catalytic subunit of PI3K were substituted with a point mutation (D910A), which is a catalytically inactive form of p110δ (p110δ^D910A), have been described (Kelly et al., 2007; Kurts et al., 1996; Pircher et al., 1989; Waugh et al., 2009). Mice were produced in the Biological Resource Unit at the University of Dundee in compliance with UK Home Office Animals (Scientific Procedures) Act 1986 guidelines.

Cell Culture

CD8⁺ T cells were isolated from spleens and/or lymph nodes with an AutoMACs magnetic cell sorter (Miltenyi Biotec). Lymphocytes were suspended in RPMI 1640 plus 10% heat-inactivated fetal calf serum (FCS), penicillin, streptomycin (GIBCO), and 50 μM β-mercaptoethanol (β-ME) (Sigma) and activated for 48 hr in either 0.5 μg/ml CD3 antibody (2C11) or 100 ng/ml LCMV TCR-specific peptide gp33-41, KAVYNFATM. Cells were then washed free of activating agent and thereafter maintained in 20 ng/ml IL-2 at a density of approximately 0.3 × 10⁶/ml at 37°C, 5% CO₂. 4-hydroxytamoxifen (4OHT, Sigma) was used at 0.6 μM. AktI-1/2 (AktI) (Calbiochem) was used at 1 μM, the p110δ inhibitor IC87114 (made in-house) and LY294002 (Promega) were used at 10 μM. Where indicated cells were cultured in media of defined glucose concentrations with glucose-free RPMI containing L-glutamine (GIBCO) with 10% dialyzed FCS (GIBCO), penicillin, streptomycin, 50 μM β-ME (glucose-free media) plus D(+)glucose (Sigma).Pdpk1-deleted CTL were prepared by activatingPdpk1^fl/fl TamoxCre splenocytes with 2C11 for 2 days and then culturing the cells in IL-2 for 5 days, with 0.6 μM 4OHT being added for the final 72 hr of culture.

Flow Cytometry

Cells were labeled in RPMI plus 0.5% FCS with Phycoerythrin-conjugated anti-CD62L, Alexa750-conjugated anti-CD4, PerCP.Cy5.5-conjugated anti-TCR Vα2, allophycocyanin-conjugated anti-CD45.2, Horizon V450-conjugated anti-CD45.1, and phycoerythrin.Cy7-conjugated anti-CD8. For FoxoAAA, IFN-γ double-staining cells were stimulated in the presence of 3 μg/ml Golgiplug (BD), fixed and permeabilized as per manufacturer's instructions (eBioscience 00-8222-49, 00-8333-56), and then stained with Alexa488-conjugated anti-GFP (Invitrogen) plus allophycocyanin-conjugated anti-IFN-γ (BD Biosciences). Data were acquired on a FACS Calibur (BD Biosciences) and analyzed with FlowJo software (Treestar). Viable cells were gated according to their forward scatter (FSC) and side scatter (SSC) profiles. GFP^+/− cell isolation was performed with a Vantage cell sorter (BD Biosciences).

Immunoblot Analysis

Cells were lysed on ice in HEPES lysis buffer: 100 mM HEPES (pH 7.4), 150 mM NaCl, 20 mM NaF, 20 mM iodoacetamide, 2 mM EDTA, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 40 mM β-glycerophosphate (Sigma), and protease inhibitors (Roche). Lysates were centrifuged (4°C, 1600 × g, 15 min) and then samples separated via sodium dodecyl sulfate 4%–12% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed for pT24 Foxo1 + p32Foxo3a; pT308 and total Akt; pT235 + pT236 and total small ribosomal subunit S6; pS389, pS421 + pS424, and total S6K; PKCθ (all Cell Signaling Technologies); pS227 p90RSK (Santa-Cruz Biotechnology); total Foxo3a (made in house); PDK1 (Upstate); or perforin (gift of G. Griffith).

Glucose and Phenylalanine Uptake

1 × 10⁶ cells were suspended in 500 μl glucose-free media for 10 min. 500 μl glucose-free media containing 1 μCi/ml 2-Deoxy-D-[1-³H]glucose ([³H] 2DOG) (GE healthcare) was then added and the cells incubated for a further 10 min. Cells were pelleted, washed in PBS, and then lysed in water. Lysate³H content was then measured via liquid scintillation counting. For phenylalanine uptake, the assay was performed essentially as for glucose uptake, except that assays were performed in HBSS, 50 μM β-ME, 1 × MEM vitamins (GIBCO), with 10% dialyzed FCS. [³H]Phe (GE Healthcare) was added and cells incubated for 1 hr prior to pelleting.

Real-Time PCR

RNA was extracted from cells with the RNeasy RNA purification minikit (QIAGEN). Reverse-transcription PCR was performed with qScript cDNA synthesis kit (Quanta). Real-time PCR was performed with iQ SYBR Green detection chemistry (BioRad) on an iCycler (BioRad). Relative mRNA levels of genes of interest were normalized to hypoxanthine guanine phosphoribosyltransferase (HPRT). Primers are detailed in theSupplemental Experimental Procedures.

ChIP

Real-time PCR-based chromatin immunoprecipitation (ChIP) analysis to measure RNA Pol II binding to the IFN-γ locus was performed described (Cruz-Guilloty et al., 2009) with minor modifications. Chromatin was immunoprecipitated with anti-Pol II (Santa-Cruz Biotechnology) or normal rabbit IgG (Cell Signaling) from 5 × 10⁶ cells in presence of 0.2 mg/ml BSA. ChIP Grade Protein G Magnetic Beads (Cell Signaling) were used to collect the immunocomplexes. Chromatin was purified via a NucleoSpin Extract II kit (Macherey-Nagel) and resuspended in TE buffer. Real-time PCR was performed in a Biorad iQ5 with Perfecta SYBR green FastMix for iQ (Quanta BioSciences). Primers are detailed in theSupplemental Experimental Procedures.

Retroviral Transduction

The production of FoxoAAA-GFP (Foxo3a T32A S252A S314A) and control retrovirus has been described previously (Waugh et al., 2009). The retroviral infection protocol was performed as described previously (Waugh et al., 2009).

IFN-γ Quantification

IFN-γ was assayed in culture supernatants with the femto-HS high-sensitivity IFN-γ ELISA kit (eBiosciences).

Microarray Analysis

Microarray analysis was carried out by the Finnish DNA Microarray Centre at the Centre for Biotechnology, Turku, Finland via 430_2.0 mouse expression arrays (Affymetrix) and the manufacturer's recommended protocol. Statistically significant differences in gene expression were identified with Multiple Experiment Viewer v4.3 (Saeed et al., 2003). Overrepresented gene ontology terms were identified with the NIAID DAVID website (http://www.david.abcc.ncifcrf.gov). Full details are given inSupplemental Experimental Procedures.

Statistical Analyses

With the exception of the microarray data, statistical analyses were performed with Prism 4.00 for Macintosh (GraphPad). A nonparametric Mann Whitney test was used where the number of experiments performed was not sufficient to prove normal distribution. Statistically significant results (p < 0.05) are indicated by an asterisk.

1. Ahmed R., Bevan M.J., Reiner S.L., Fearon D.T. The precursors of memory: models and controversies. Nat. Rev. Immunol. 2009;9:662–668. doi: 10.1038/nri2619. [DOI] [PubMed] [Google Scholar]
2. Araki K., Turner A.P., Shaffer V.O., Gangappa S., Keller S.A., Bachmann M.F., Larsen C.P., Ahmed R. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009;460:108–112. doi: 10.1038/nature08155. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Bachmann M.F., Wolint P., Walton S., Schwarz K., Oxenius A. Differential role of IL-2R signaling for CD8+ T cell responses in acute and chronic viral infections. Eur. J. Immunol. 2007;37:1502–1512. doi: 10.1002/eji.200637023. [DOI] [PubMed] [Google Scholar]
4. Bain J., Plater L., Elliott M., Shpiro N., Hastie C.J., McLauchlan H., Klevernic I., Arthur J.S., Alessi D.R., Cohen P. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 2007;408:297–315. doi: 10.1042/BJ20070797. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Bayascas J.R. Dissecting the role of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) signalling pathways. Cell Cycle. 2008;7:2978–2982. doi: 10.4161/cc.7.19.6810. [DOI] [PubMed] [Google Scholar]
6. Bayascas J.R., Wullschleger S., Sakamoto K., García-Martínez J.M., Clacher C., Komander D., van Aalten D.M., Boini K.M., Lang F., Lipina C. Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance. Mol. Cell. Biol. 2008;28:3258–3272. doi: 10.1128/MCB.02032-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Carlson C.M., Endrizzi B.T., Wu J., Ding X., Weinreich M.A., Walsh E.R., Wani M.A., Lingrel J.B., Hogquist K.A., Jameson S.C. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature. 2006;442:299–302. doi: 10.1038/nature04882. [DOI] [PubMed] [Google Scholar]
8. Castellino F., Germain R.N. Chemokine-guided CD4+ T cell help enhances generation of IL-6RalphahighIL-7Ralpha high prememory CD8+ T cells. J. Immunol. 2007;178:778–787. doi: 10.4049/jimmunol.178.2.778. [DOI] [PubMed] [Google Scholar]
9. Cham C.M., Driessens G., O'Keefe J.P., Gajewski T.F. Glucose deprivation inhibits multiple key gene expression events and effector functions in CD8+ T cells. Eur. J. Immunol. 2008;38:2438–2450. doi: 10.1002/eji.200838289. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Cornish G.H., Sinclair L.V., Cantrell D.A. Differential regulation of T-cell growth by IL-2 and IL-15. Blood. 2006;108:600–608. doi: 10.1182/blood-2005-12-4827. [DOI] [PubMed] [Google Scholar]
11. Cruz-Guilloty F., Pipkin M.E., Djuretic I.M., Levanon D., Lotem J., Lichtenheld M.G., Groner Y., Rao A. Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs. J. Exp. Med. 2009;206:51–59. doi: 10.1084/jem.20081242. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. D'Souza W.N., Lefrançois L. IL-2 is not required for the initiation of CD8 T cell cycling but sustains expansion. J. Immunol. 2003;171:5727–5735. doi: 10.4049/jimmunol.171.11.5727. [DOI] [PubMed] [Google Scholar]
13. Fabre S., Carrette F., Chen J., Lang V., Semichon M., Denoyelle C., Lazar V., Cagnard N., Dubart-Kupperschmitt A., Mangeney M. FOXO1 regulates L-Selectin and a network of human T cell homing molecules downstream of phosphatidylinositol 3-kinase. J. Immunol. 2008;181:2980–2989. doi: 10.4049/jimmunol.181.5.2980. [DOI] [PubMed] [Google Scholar]
14. Finlay D.K., Sinclair L.V., Feijoo C., Waugh C.M., Hagenbeek T.J., Spits H., Cantrell D.A. Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes. J. Exp. Med. 2009;206:2441–2454. doi: 10.1084/jem.20090219. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Fox C.J., Hammerman P.S., Thompson C.B. The Pim kinases control rapamycin-resistant T cell survival and activation. J. Exp. Med. 2005;201:259–266. doi: 10.1084/jem.20042020. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Garçon F., Patton D.T., Emery J.L., Hirsch E., Rottapel R., Sasaki T., Okkenhaug K. CD28 provides T-cell costimulation and enhances PI3K activity at the immune synapse independently of its capacity to interact with the p85/p110 heterodimer. Blood. 2008;111:1464–1471. doi: 10.1182/blood-2007-08-108050. [DOI] [PubMed] [Google Scholar]
17. Green C.J., Göransson O., Kular G.S., Leslie N.R., Gray A., Alessi D.R., Sakamoto K., Hundal H.S. Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake. J. Biol. Chem. 2008;283:27653–27667. doi: 10.1074/jbc.M802623200. [DOI] [PubMed] [Google Scholar]
18. Hand T.W., Cui W., Jung Y.W., Sefik E., Joshi N.S., Chandele A., Liu Y., Kaech S.M. Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival. Proc. Natl. Acad. Sci. USA. 2010;107:16601–16606. doi: 10.1073/pnas.1003457107. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Hendriks J., Xiao Y., Borst J. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J. Exp. Med. 2003;198:1369–1380. doi: 10.1084/jem.20030916. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Jacobs S.R., Herman C.E., Maciver N.J., Wofford J.A., Wieman H.L., Hammen J.J., Rathmell J.C. Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways. J. Immunol. 2008;180:4476–4486. doi: 10.4049/jimmunol.180.7.4476. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Janas M.L., Groves P., Kienzle N., Kelso A. IL-2 regulates perforin and granzyme gene expression in CD8+ T cells independently of its effects on survival and proliferation. J. Immunol. 2005;175:8003–8010. doi: 10.4049/jimmunol.175.12.8003. [DOI] [PubMed] [Google Scholar]
22. Jarmin S.J., David R., Ma L., Chai J.G., Dewchand H., Takesono A., Ridley A.J., Okkenhaug K., Marelli-Berg F.M. T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice. J. Clin. Invest. 2008;118:1154–1164. doi: 10.1172/JCI33267. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Juntilla M.M., Wofford J.A., Birnbaum M.J., Rathmell J.C., Koretzky G.A. Akt1 and Akt2 are required for alphabeta thymocyte survival and differentiation. Proc. Natl. Acad. Sci. USA. 2007;104:12105–12110. doi: 10.1073/pnas.0705285104. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Kaech S.M., Tan J.T., Wherry E.J., Konieczny B.T., Surh C.D., Ahmed R. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 2003;4:1191–1198. doi: 10.1038/ni1009. [DOI] [PubMed] [Google Scholar]
25. Kalia V., Sarkar S., Subramaniam S., Haining W.N., Smith K.A., Ahmed R. Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity. 2010;32:91–103. doi: 10.1016/j.immuni.2009.11.010. [DOI] [PubMed] [Google Scholar]
26. Kelly A.P., Finlay D.K., Hinton H.J., Clarke R.G., Fiorini E., Radtke F., Cantrell D.A. Notch-induced T cell development requires phosphoinositide-dependent kinase 1. EMBO J. 2007;26:3441–3450. doi: 10.1038/sj.emboj.7601761. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Kerdiles Y.M., Beisner D.R., Tinoco R., Dejean A.S., Castrillon D.H., DePinho R.A., Hedrick S.M. Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. Nat. Immunol. 2009;10:176–184. doi: 10.1038/ni.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Kurts C., Heath W.R., Carbone F.R., Allison J., Miller J.F., Kosaka H. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 1996;184:923–930. doi: 10.1084/jem.184.3.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Lin J.X., Spolski R., Leonard W.J. Critical role for Rsk2 in T-lymphocyte activation. Blood. 2008;111:525–533. doi: 10.1182/blood-2007-02-072207. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Liu D., Uzonna J.E. The p110 delta isoform of phosphatidylinositol 3-kinase controls the quality of secondary anti-Leishmania immunity by regulating expansion and effector function of memory T cell subsets. J. Immunol. 2010;184:3098–3105. doi: 10.4049/jimmunol.0903177. [DOI] [PubMed] [Google Scholar]
31. Maciver N.J., Jacobs S.R., Wieman H.L., Wofford J.A., Coloff J.L., Rathmell J.C. Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival. J. Leukoc. Biol. 2008;84:949–957. doi: 10.1189/jlb.0108024. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Malek T.R., Castro I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity. 2010;33:153–165. doi: 10.1016/j.immuni.2010.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Manjunath N., Shankar P., Wan J., Weninger W., Crowley M.A., Hieshima K., Springer T.A., Fan X., Shen H., Lieberman J., von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 2001;108:871–878. doi: 10.1172/JCI13296. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Mao C., Tili E.G., Dose M., Haks M.C., Bear S.E., Maroulakou I., Horie K., Gaitanaris G.A., Fidanza V., Ludwig T. Unequal contribution of Akt isoforms in the double-negative to double-positive thymocyte transition. J. Immunol. 2007;178:5443–5453. doi: 10.4049/jimmunol.178.9.5443. [DOI] [PubMed] [Google Scholar]
35. Obar J.J., Molloy M.J., Jellison E.R., Stoklasek T.A., Zhang W., Usherwood E.J., Lefrançois L. CD4+ T cell regulation of CD25 expression controls development of short-lived effector CD8+ T cells in primary and secondary responses. Proc. Natl. Acad. Sci. USA. 2010;107:193–198. doi: 10.1073/pnas.0909945107. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Ouyang W., Beckett O., Flavell R.A., Li M.O. An essential role of the Forkhead-box transcription factor Foxo1 in control of T cell homeostasis and tolerance. Immunity. 2009;30:358–371. doi: 10.1016/j.immuni.2009.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Pearce E.L., Walsh M.C., Cejas P.J., Harms G.M., Shen H., Wang L.S., Jones R.G., Choi Y. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature. 2009;460:103–107. doi: 10.1038/nature08097. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Pipkin M.E., Sacks J.A., Cruz-Guilloty F., Lichtenheld M.G., Bevan M.J., Rao A. Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Immunity. 2010;32:79–90. doi: 10.1016/j.immuni.2009.11.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Pircher H., Bürki K., Lang R., Hengartner H., Zinkernagel R.M. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature. 1989;342:559–561. doi: 10.1038/342559a0. [DOI] [PubMed] [Google Scholar]
40. Prlic M., Bevan M.J. Immunology: A metabolic switch to memory. Nature. 2009;460:41–42. doi: 10.1038/460041a. [DOI] [PubMed] [Google Scholar]
41. Rao R.R., Li Q., Odunsi K., Shrikant P.A. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin. Immunity. 2010;32:67–78. doi: 10.1016/j.immuni.2009.10.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Rathmell J.C., Elstrom R.L., Cinalli R.M., Thompson C.B. Activated Akt promotes increased resting T cell size, CD28-independent T cell growth, and development of autoimmunity and lymphoma. Eur. J. Immunol. 2003;33:2223–2232. doi: 10.1002/eji.200324048. [DOI] [PubMed] [Google Scholar]
43. Saeed A.I., Sharov V., White J., Li J., Liang W., Bhagabati N., Braisted J., Klapa M., Currier T., Thiagarajan M. TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003;34:374–378. doi: 10.2144/03342mt01. [DOI] [PubMed] [Google Scholar]
44. Saibil S.D., Jones R.G., Deenick E.K., Liadis N., Elford A.R., Vainberg M.G., Baerg H., Woodgett J.R., Gerondakis S., Ohashi P.S. CD4+ and CD8+ T cell survival is regulated differentially by protein kinase Ctheta, c-Rel, and protein kinase B. J. Immunol. 2007;178:2932–2939. doi: 10.4049/jimmunol.178.5.2932. [DOI] [PubMed] [Google Scholar]
45. Sinclair L.V., Finlay D., Feijoo C., Cornish G.H., Gray A., Ager A., Okkenhaug K., Hagenbeek T.J., Spits H., Cantrell D.A. Phosphatidylinositol-3-OH kinase and nutrient-sensing mTOR pathways control T lymphocyte trafficking. Nat. Immunol. 2008;9:513–521. doi: 10.1038/ni.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Soond D.R., Bjørgo E., Moltu K., Dale V.Q., Patton D.T., Torgersen K.M., Galleway F., Twomey B., Clark J., Gaston J.S. PI3K p110delta regulates T-cell cytokine production during primary and secondary immune responses in mice and humans. Blood. 2010;115:2203–2213. doi: 10.1182/blood-2009-07-232330. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Tamás P., Macintyre A., Finlay D., Clarke R., Feijoo-Carnero C., Ashworth A., Cantrell D. LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells. Eur. J. Immunol. 2010;40:242–253. doi: 10.1002/eji.200939677. [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Waugh C., Sinclair L., Finlay D., Bayascas J.R., Cantrell D. Phosphoinositide (3,4,5)-triphosphate binding to phosphoinositide-dependent kinase 1 regulates a protein kinase B/Akt signaling threshold that dictates T-cell migration, not proliferation. Mol. Cell. Biol. 2009;29:5952–5962. doi: 10.1128/MCB.00585-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Williams M.R., Arthur J.S., Balendran A., van der Kaay J., Poli V., Cohen P., Alessi D.R. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr. Biol. 2000;10:439–448. doi: 10.1016/s0960-9822(00)00441-3. [DOI] [PubMed] [Google Scholar]
50. Williams M.A., Tyznik A.J., Bevan M.J. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature. 2006;441:890–893. doi: 10.1038/nature04790. [DOI] [PMC free article] [PubMed] [Google Scholar]

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