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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Immunity. 2013 Dec 5;39(6):1043–1056. doi: 10.1016/j.immuni.2013.09.015

T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic programming

Kai Yang 1, Sharad Shrestha 1, Hu Zeng 1, Peer WF Karmaus 1, Geoffrey Neale 2, Peter Vogel 3, David A Guertin 4, Richard F Lamb 5, Hongbo Chi 1
PMCID: PMC3986063  NIHMSID: NIHMS542075  PMID: 24315998

SUMMARY

Naïve T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic programming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and Th2 cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 co-stimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity.

Keywords: quiescence, immune response, T cell metabolism, Th2 cell differentiation, co-stimulation

INTRODUCTION

Mature T cells circulate through peripheral lymphoid organs in a quiescent state (G0) characterized by small cell size and low metabolic activity (Hamilton and Jameson, 2012). Antigen stimulation through T cell receptors (TCRs) activates T cells, leading to clonal expansion and effector differentiation. Associated with T cell activation is a marked increase of metabolic activities such as glycolysis (Wang and Green, 2012). Despite extensive knowledge on early TCR signaling (e.g. NF-κB and MAPK activation) and subsequent clonal expansion and differentiation, how these processes are connected remains unclear. One notable feature of this transitional period is the extended time required (>24 h), as compared with subsequent cell divisions that involve rapid doubling (every 5–6 h) (Rowell and Wells, 2006). However, little is understood about the mechanisms whereby naïve T cells transit from quiescence to the entry of the first cell cycle, or the extent to which this transitional process impacts ensuing clonal expansion and functional differentiation.

The mechanistic target of rapamycin (mTOR) pathway is a crucial regulator of T cell responses (Chi, 2012). mTOR signaling is comprised of mTORC1 and mTORC2 complexes defined by the signature components Raptor and Rictor, respectively. Blocking mTORC1 by rapamycin exerts anti-proliferative and other immune suppressive effects. However, rapamycin-resistant proliferation has been observed in various T cell populations (Finlay et al., 2012; Fox et al., 2005), and rapamycin can even promote immune responses in select conditions (Araki et al., 2009). Notably, rapamycin can block mTORC2 activity upon prolonged or high-dose treatment in CD4+ T cells (Delgoffe et al., 2011) but not in effector CD8+ T cells (Finlay et al., 2012).

Additionally, rapamycin is not an efficient inhibitor of mTORC1-mediated 4E-BP1 phosphorylation (Choo et al., 2008). Moreover, because of the broad effects of rapamycin on multiple cell types, use of rapamycin in vivo is unlikely to reveal T cell-intrinsic requirement of mTOR. Instead, T cell-specific deletion systems have been instrumental in dissecting the specific roles of mTOR in T cell responses. In CD4+ T cells, loss of Rheb, an important upstream activator of mTORC1, inhibits the differentiation of Th1 and Th17 effector cells (Delgoffe et al., 2009; Delgoffe et al., 2011), whereas deletion of Raptor impairs Th17 cell differentiation (Kurebayashi et al., 2012). Further, Th2 cell differentiation has been shown to require mTORC2 activity (Delgoffe et al., 2011; Lee et al., 2010), independent of Rheb-dependent mTORC1 (Delgoffe et al., 2011). Finally, T cells lacking Rheb exhibit modestly reduced proliferation and normal IL-2 production that suggest a limited role of mTORC1 in early T cell priming (Delgoffe et al., 2011). However, it is important to note that multiple upstream inputs feed into mTORC1, some of which are independent of Rheb or PI3K-AKT (Finlay et al., 2012; Gwinn et al., 2008). Also, Rheb has non-conventional activities independently of mTORC1 (Neuman and Henske, 2011), highlighting the complexity of mTORC1 regulation. Furthermore, although the metabolic function of mTORC1 is well recognized (Duvel et al., 2010), little is understood how this is regulated in T cells (Zeng and Chi, 2013). Altogether, the physiological significance and mechanistic basis of mTORC1 in T cell functions remain controversial and unclear.

Capitalizing on genetic deletion of Raptor, here we report that mTORC1 is a central regulator of adaptive immunity. Among components of mTOR signaling tested, Raptor has a predominant role in regulating T cell priming and in vivo immune responses, whereas Rictor-mTORC2 and Rheb exert more modest effects. Mechanistically, Raptor-mTORC1 orchestrates the glycolytic and lipogenic programs to drive the exit of naïve T cells from the quiescent G0 state. Further, Raptor-mediated metabolic reprogramming plays a central role in instructing Th2 cell differentiation, by integrating TCR and CD28 signals and coupling them to cytokine responsiveness. Our studies identify a Raptor-mTORC1-mediated pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates cell proliferation and fate decisions.

RESULTS

Raptor deletion impairs T cell activation and proliferation

To investigate the roles of Raptor in T cell functions, we crossed mice with loxP-flanked Rptor alleles (Rptorfl/fl) with CD4-Cre mice to delete Rptor specifically in T cells (called ‘Rptor−/−’; Figures S1A,B). Wild-type (WT) and Rptor−/− mice had similar numbers of total thymocytes and major subsets (Figure S1C). Peripheral CD4+ T cells in Rptor−/− mice were undisturbed, whereas CD8+ T cells showed a modest reduction in the spleen (Figure 1A) and lymph nodes (data not shown). Further, Rptor−/− mice contained a reduced CD62LloCD44hi population with an activation or memory phenotype, especially in CD8+ cells (Figure 1B). Thus, Raptor contributes to homeostasis of peripheral T cells, especially CD8+ cells, under steady state.

Figure 1. Raptor is required for T cell activation and proliferation in vitro and in vivo.

Figure 1

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(A) Flow cytometry of CD4+ and CD8+ T cells in the spleens of WT and Rptor−/− mice. Right panels, proportions and numbers of CD4+ and CD8+ T cells (n=3 mice per group). (B) Expression of CD62L and CD44 on CD4+ and CD8+ T cells. (C,D) IL-2 secretion (C) and mRNA expression (D) in CD4+ T cells stimulated with α -CD3-CD28. (E) [3H]Thymidine incorporation of CD4+ T cells stimulated with α-CD3 or α-CD3-CD28 for 64 h and pulsed with [3H]thymidine for an additional 8 h. (F) Flow cytometry of CFSE-labeled CD4+ T cells stimulated with α-CD3-CD28 for 72 h. (G,H) CFSE-labeled WT OT-II or Rptor−/− OT-II T cells (CD45.2+) were transferred into CD45.1+ recipients and immunized s.c. with OVA323–339 in CFA 24 h later, followed by analysis after an additional 3 d. G, Proportion and number of donor OT-II T cells in the draining lymph nodes of recipients (n=3 mice per group). H, Flow cytometry of CFSE (left) and cell size (right) of donor OT-II T cells. (I,J) CFSE-labeled CD4+ T cells were transferred into sublethally irradiated CD45.1+ (I) or non-irradiated Rag1−/− mice (J), followed by analysis at day 7 after transfer. Data are representative of 2 (A-D,G-J) or 3 (E,F) independent experiments, and error bars represent the SEM. See also Figure S1.

Antigen stimulation induces activation and clonal expansion of naïve T cells. We analyzed TCR-induced initial activation and ensuing proliferation in Rptor−/− naïve CD4+ T cells (CD62LhiCD44loCD25−; unless otherwise noted, naïve T cells were used throughout this study). IL-2 production, a hallmark of T cell activation, was substantially reduced in Rptor−/− T cells stimulated with TCR and CD28 (Figures 1C,D). Following stimulation with α-CD3 with or without α-CD28, WT cells underwent proliferation as measured by [3H]thymidine incorporation. However, greatly reduced proliferation was observed in Rptor−/− CD4+ (Figure 1E) and CD8+ T cells (Figure S1D). To distinguish the contribution from cell death and division, we measured these two effects specifically by tracking incorporation of propidium iodide (PI) and dilution of CFSE. WT and Rptor−/− T cells showed comparable survival in the presence or absence of TCR stimulation (Figure S1E). In contrast, whereas CFSE-labeled WT cells underwent robust division after 72 h of TCR stimulation, the division of Rptor−/− T cells was markedly reduced (Figure 1F). Furthermore, the proliferative defect of Rptor−/− T cells was not secondary to impaired IL-2 production, as exogenous IL-2 did not restore the proliferation (Figure S1F). These results establish the crucial function of Raptor in TCR-induced activation and proliferation in vitro.

To address the role of Raptor in vivo, we crossed Raptor deficiency onto the OT-II TCR-transgenic mouse model, which allowed us to visualize proliferation of antigen-specific T cells in vivo. We sorted naïve T cells from WT OT-II and Rptor−/− OT-II mice, labeled them with CFSE, and transferred them into congenic CD45.1+ mice, followed by s.c. immunization with OVA323–339 peptide. At 72 h following immunization, Rptor−/− OT-II donor cells showed much less accumulation in the recipients’ draining lymph nodes (Figure 1G), associated with markedly reduced CFSE dilution and cell growth (Figure 1H). Therefore, Raptor function is crucial for antigen-induced T cell proliferation in vivo.

Aside from antigen-induced proliferation, transfer of naïve T cells into lymphopenic environment also initiates rapid proliferation. Such proliferative responses involve distinct metabolic requirements from antigen-specific proliferation (Kidani et al., 2013; Sena et al., 2013), and can be further divided into homeostatic and spontaneous proliferation (Kieper et al., 2005; Min et al., 2005). To determine the role of Raptor in lymphopenia-induced proliferation, we transferred CFSE-labeled naïve T cells into sublethally irradiated CD45.1+ or unmanipulated Rag1−/− mice to trigger homeostatic or spontaneous proliferation, respectively (Kieper et al., 2005; Min et al., 2005). Rptor−/− donor T cells showed marked defects of proliferation in both types of recipients (Figures 1I,J). Notably, Raptor deletion did not affect cell survival under steady state (data not shown) or upon in vitro stimulation with IL-7 (Figure S1G). These findings collectively indicate that Raptor is essential for both antigen-specific and lymphopenia-induced proliferation.

A central role of Raptor, but not Rictor, in T cell priming

To determine the role of Raptor in immune responses in vivo, we infected WT and Rptor−/− mice with recombinant Listeria monocytogenes expressing ovalbumin (OVA). CD4+ T cells from infected Rptor−/− mice contained a greatly reduced proportion of antigen-specific IFN-γ+ cells following restimulation with LLO189–201 peptide (Figures 2A,B). Rptor−/− mice also had markedly reduced IFN-γ+ CD8+ T cells after OVA257–264 restimulation (Figures 2C,D). Additionally, CD8+ T cells from infected Rptor−/− mice had significantly fewer tetramer-positive cells than did those from controls (Figures S2A,B). Thus, Raptor deletion essentially abolished anti-bacterial immune responses in vivo.

Figure 2. Deficiency of Raptor but not Rictor markedly impairs antigen-specific immune responses in vivo and in vitro.

Figure 2

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(A,B) Flow cytometry of OVA-reactive IFN- γ+ CD4+ T cells from mice infected with OVA-expressing L. monocytogenes, detected after LLO189–201 stimulation and intracellular cytokine staining (A). B, proportion and number of LLO-reactive IFN-γ+ CD4+ T cells. (C,D) Flow cytometry of OVA-reactive IFN-γ+ CD8+ T cells from infected mice, detected after OVA257–264 stimulation and intracellular cytokine staining (C). D, proportion and number of OVA-reactive IFN-γ+ CD8+ T cells. (E) [3H]Thymidine incorporation of CD4+ T cells stimulated with α-CD3 or α-CD3-CD28 for 64 h, and pulsed with [3H]thymidine for an additional 8 h. Data are combined results from 3 (A–D) independent or representative of 2 (E) independent experiments, and error bars represent the SEM. See also Figure S2.

Given the frequent cross-regulation of mTORC1 and mTORC2 activities (Zoncu et al., 2011), we examined the effect of mTORC2 in anti-Listeria immune responses by analyzing mice with CD4-Cre-mediated deletion of Rictor to ablate mTORC2 activity (Rictorfl/flCD4-Cre (Yang et al., 2011), called ‘Rictor−/−’), and Rptor−/−Rictor−/− mice. Rictor−/− mice showed a small reduction in the generation of IFN-γ+ cells (Figures 2A–D), as well as in the expansion of tetramer-positive T cells (Figures S2A,B). Antigen-specific responses were blunted in mice deficient in both mTOR complexes, and these effects were largely comparable to the loss of Raptor alone (Figures 2A–D, S2A,B). Therefore, Raptor clearly plays a predominant role in anti-bacterial immune responses.

We next compared the effects of Raptor and Rictor loss on T cell proliferation in vitro. In contrast to the severely impaired proliferation of Rptor−/− T cells, the proliferative defect of Rictor−/− T cells was less profound especially when stimulated with optimal α-CD3-CD28 antibodies (Delgoffe et al., 2011; Lee et al., 2010) (Figure 2E). Similar results were observed in antigen-specific OT-II T cells (Figure S2C). Further, Rptor−/−Rictor−/− and Rptor−/− T cells showed a comparable defect in proliferation (Figure 2E). Thus, in vivo priming and in vitro proliferation of T cells have a more stringent requirement of Raptor than Rictor function.

Preferential requirement of Raptor for cell cycle entry from quiescence

We next determined the specific stage in cell proliferation that requires Raptor-mTORC1 function. When T cells were stimulated with α-CD3-CD28 for 24 h and pulse-labeled with BrdU, over 20% of WT cells incorporated BrdU. However, less than 1% of Rptor−/− cells accomplished this (Figure 3A), indicating a defect to enter S phase. TCR stimulation also results in increased cell size, namely cell growth, which is required for cell cycle entry (Lea et al., 2003). Freshly isolated Rptor−/− naïve T cells showed a slightly reduced cell size (Figure S3A). After TCR stimulation for 18 h, WT cells were substantially enlarged, but size increase in Rptor−/− T cells was much less pronounced (Figure S3A). Cell growth is dependent on the regulated expression of amino-acid transporters that include CD98 as a key component, and the transferrin receptor CD71 for iron uptake. Although expression of these receptors is inhibited by rapamycin (Edinger and Thompson, 2002), the relative contributions from mTORC1 and mTORC2 are unclear. Rptor−/− T cells showed marked defects in TCR-induced CD98 and CD71 expression, whereas Rictor−/− T cells exhibited no major defects (Figure 3B). These data reveal a key role of Raptor in cell growth and nutrient uptake that may contribute to cell cycle entry.

Figure 3. Raptor-mTORC1 signaling is mainly required for cell cycle entry from quiescence instead of continuous proliferation.

Figure 3

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(A) BrdU staining of CD4+ T cells stimulated with α -CD3-CD28 for 22 h, followed by pulsing with BrdU for 2 h. (B) Expression of CD71 and CD98 on CD4+ T cells stimulated with α-CD3-CD28 for 18 h. (C) Scatter plot comparing global gene expression profiles between WT and Rptor−/− CD4+ T cells stimulated with α-CD3-CD28 for 24 h (n=4 mice per group). Probe sets containing the cell cycle genes with twofold or greater difference between WT and Rptor−/−T cells are shown in red, with select genes annotated. mRNA expression levels are on a log2 scale. (D) Expression of Raptor, cyclin D2, cyclin E, CDK2, CDK4, and CDK6 in CD4+ T cells stimulated with α-CD3-CD28. (E) Expression and phosphorylation of Rb in CD4+ T cells stimulated with α-CD3-CD28. (F) CFSE-labeled CD4+ T cells were stimulated with α-CD3-CD28 for 70 h, pulsed with BrdU for 2 h, and subjected to 7-AAD and BrdU staining. Presented is BrdU staining of the gated CFSE-diluted population. (G) WT and Rptorfl/fl CD4+ T cells were infected with EGFP-Cre retrovirus at 24 h after α-CD3-CD28 stimulation, and 4 d later, GFP+ cells were sorted and stimulated with α-CD3-CD28 for 24 h for BrdU incorporation assay. (H) WT CD4+ T cells were stimulated with α-CD3-CD28, and rapamycin was added at different time points (left panel). Cells were subjected to BrdU staining at 24 h after addition of rapamycin. Data are representative of 3 (A,B,H), 1 (C; n=4 mice per group in the microarrays) or 2 (D–G) independent experiments. See also Figure S3 and Tables S1 and S2.

To understand the mechanistic basis, we performed bioinformatic analysis to identify Raptor-dependent pathways in TCR and CD28-stimulated cells at 0, 8 and 24 h. Remarkably, out of the 901 probes (representing 594 individual genes) with twofold or greater difference (with false discovery rate, FDR<0.05) at 24 h, 212 probes (128 genes) were associated with cell cycle regulation and were downregulated in Rptor−/− cells (Figure 3C and Table S1). Next, to identify key networks regulated by Raptor, we performed gene set enrichment analysis (GESA) to compare the transcriptomes of WT and Rptor−/− T cells without arbitrary cut-offs (Subramanian et al., 2005). This unbiased analysis revealed that the cell cycle-related pathways constituted the top 10 most significantly downregulated gene sets in Rptor−/− T cells at 24 h of stimulation (data not shown), with the cell cycle mitotic pathway as the top hit (Normalized Enrichment Score, NES=−3.25, FDR<0.001; Figure S3B). To dissect detailed kinetics of Raptor-dependent cell cycle entry, we also analyzed gene expression profiles at 0 and 8 h of TCR and CD28 stimulation. Whereas very few probes (46 in total) were differentially expressed before activation (data not shown), a total of 202 probes (126 genes) showed equal or greater than twofold change (FDR<0.05) between WT and Rptor−/− T cells after 8 h of stimulation (Table S2). Although fewer cell cycle genes were altered at 8 h by the twofold cut-off, GSEA analysis revealed that the genes involved in M/G1 transition of cell cycle were significantly reduced in Rptor−/− T cells (FDR=−2.84, FDR<0.001; Figure S3C). We next directly examined whether Raptor loss affects the cell cycle machinery. TCR and CD28 stimulation of WT CD4+ T cells induced the expression of cyclins D2 and E, and CDK2, 4 and 6, but this induction was diminished in Rptor−/− cells (Figure 3D). Further, cyclin-dependent phosphorylation of the cell cycle regulator Rb, specifically at Ser807 and Ser811 (mediated by cyclin D–CDK4 or CDK6) and Thr821 (cyclin E–CDK2), occurred after TCR stimulation in WT cells but was greatly reduced in Rptor−/− T cells (Figure 3E). Thus, Raptor deficiency impairs initiation of the cell cycle machinery in TCR-signaled cells.

Having established a role of Raptor in cell cycle entry of naïve cells, we next tested whether Raptor was also required for the proliferation of activated effector T cells. In contrary to naïve T cells that respond to antigens by entering cell cycle from quiescence (G0), activated T cells continuously proliferate and enter cell cycle from G1 phase without returning to quiescence (Rowell and Wells, 2006). We first stimulated CFSE-labeled T cells with TCR ligation for 3 d, and then analyzed CFSE-diluted T cells, which represented cells that had entered cell cycle, for BrdU incorporation. Although Rptor−/− CD4+ T cells showed less CFSE dilution, those that had diluted CFSE showed comparable BrdU incorporation as WT counterparts (Figure 3F). Second, we activated Rptor+/+ and Rptorfl/fl CD4+ T cells (lacking the CD4-Cre transgene) to induce cell cycle entry, and then deleted Raptor by transducing the cells with a retroviral vector expressing Cre linked to EGFP (Figure S3D). We observed a similar degree of BrdU incorporation of Rptor+/+EGFP-Cre+ and Rptorfl/flEGFP-Cre+ T cells stimulated with α-CD3-CD28 for 24 h (Figure 3G). These results indicate that once T cells enter cell cycle, Raptor is not essential for their continuous proliferation.

We therefore hypothesized that mTORC1 mainly regulates the exit from quiescence of naïve T cells, rather than cell cycle progression of activated cells. To test this idea, we inhibited mTORC1 via rapamycin at different time points and performed [3H]thymidine incorporation assay at 72 h of TCR stimulation. Rapamycin strongly inhibited T cell proliferation when added at the time of TCR stimulation. However, it was much less effective when added at later time points (Figure S3E), even though it blocked S6K1 phosphorylation at all the time points examined (Figure S3F). One potential caveat with this treatment regimen was the differential durations of rapamycin in the culture. To circumvent this, we added rapamycin at different times, and measured proliferation by BrdU incorporation always at 24 h after drug treatment. Consistent with the defect in Rptor−/− cells (Figure 3A), treatment of WT cells with rapamycin at the time of TCR stimulation inhibited BrdU incorporation. In contrast, rapamycin showed a much milder effect when added at 24 h after TCR stimulation, and had no effect when added at 48 h (Figure 3H). Similar effects were observed with the more potent mTOR inhibitors Torin1 and PP242 (Benjamin et al., 2011) (Figure S3G). Altogether, Raptor-mTORC1 is critical for the transition from quiescent G0 to S phase in naïve T cells, but becomes less important once T cells have entered active cell cycle.

Raptor-mTORC1 coordinates multiple metabolic programs in T cell activation

We explored the signaling and mechanistic basis underlying Raptor functions in T cells. For TCR-induced signaling pathways, phosphorylation of S6K1, S6 and 4E–BP1, the main targets of mTORC1, was abolished in Rptor−/− cells under both long- and short-term TCR stimulation (Figures 4A,B). In contrast, TCR induced comparable phosphorylation of ERK and IκBα, whereas phosphorylation of AKT at Ser473 was modestly elevated in Rptor−/− T cells (Figure 4B), in line with the loss of mTORC1-mediated feedback inhibition of mTORC2 (Zoncu et al., 2011). These results indicate that Raptor deficiency abolishes mTORC1 activity but does not cause global defects in TCR signaling.

Figure 4. Raptor-mTORC1 signaling coordinates glycolysis, lipid genesis and oxidative phosphorylation of activated T cells.

Figure 4

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(A,B) Phosphorylation of S6K1, S6 and 4E-BP1 (A) or AKT, 4E-BP1, ERK and IκBα (B) in CD4+ T cells stimulated with α-CD3-CD28. (C) Glycolytic activity of CD4+ T cells stimulated with or without α-CD3-CD28 for 24 h. (D) Real-time PCR analysis of glycolytic genes in CD4+ T cells stimulated with α-CD3-CD28. (E,F) Immunoblot (E) and real-time PCR analysis (F) of c-Myc expression in CD4+ T cells stimulated with α-CD3-CD28. (G) De novo lipid biosynthesis of CD4+ T cells stimulated with or without α-CD3-CD28 for 24 h. (H) Expression of SREBP1 and SREBP2 full-length (Fl) and mature (M) forms in CD4+ T cells stimulated with α-CD3-CD28. Right, densitometric analysis of abundance of SREBP isoforms. (I) Real-time PCR analysis of Srebf1 and Srebf2 mRNA in CD4+ T cells stimulated with α-CD3-CD28. (J) Oxygen consumption rate (OCR) of CD4+ T cells stimulated with α-CD3-CD28 for 24 h. Data are representative of 2 independent experiments, and error bars represent the SEM. See also Figure S4.

Although mTORC1 has been implicated in metabolic activation (Duvel et al., 2010), detailed mechanisms are lacking; also, most evidence to date is derived from pharmacological inhibition. Among the plethora of metabolic pathways important for rapidly proliferating cells is glycolysis (Wang and Green, 2012). We therefore measured whether Raptor deficiency impairs TCR-induced glycolytic activity. WT CD4+ T cells markedly upregulated glycolysis after TCR and CD28 stimulation (Figure 4C). Rptor−/− T cells did not show difference from WT cells at the basal level, but TCR-induced upregulation was diminished at 24 h after stimulation (Figure 4C). This was associated with reduced mRNA expression of glycolytic enzymes including Hk2, Ldha and Tpi1 (Figure 4D). Furthermore, induction of protein but not mRNA expression of c-Myc, a crucial transcription factor for T cell glycolysis (Wang et al., 2011), was attenuated in Rptor−/−cells (Figures 4E,F). These results indicate an important role of Raptor in TCR-induced glycolysis. We next tested the functional and temporal effects of blocking glycolysis on T cell proliferation. The prototypical inhibitor of glycolysis, 2-deoxyglucose (2-DG), inhibited T cell proliferation when added to T cells at the time of activation (Figure S4A). The anti-proliferative effect of 2-DG was less obvious when added at later time points (Figure S4A), even though it potently inhibited T cell glycolytic activity at all the time points examined (Figure S4B). Further, direct numeration of cultured cells also indicated that 2-DG was more effective when added at early than late time points of TCR stimulation (Figure S4C). These results recapitulated the findings from mTOR inhibitors described above. Altogether, glucose metabolism activated by mTORC1 contributes to the transition from quiescence to proliferation.

We next performed gene ontology (GO) analysis to identify additional metabolic pathways mediated by Raptor. All of the top 10 pathways altered at 8 h of TCR stimulation (≥2 fold) were associated with the biosynthetic pathways of lipids (i.e. cholesterols and fatty acids), which were downregulated in Rptor−/− T cells (data not shown). The heat maps in Figure S4D showed the defective induction of genes involved in lipogenic pathways, many of which were verified by real-time PCR analysis (Figure S4E). These include genes in cholesterol synthesis and uptake (Hmgcs1, Hmgcr, Idi1, Sqle, Dhcr24, and Ldlr), as well as those in fatty acid synthesis, although the defects in the latter group were less profound (Fasn) or occurred only at a later time point (Scd1 and Scd2) (Figure S4E). To determine whether these changes correlated with altered de novo lipid synthesis, we measured the incorporation of [1-14C] acetate into chloroform- or methanol-soluble lipids. Rptor−/− T cells were impaired in TCR-induced de novo lipid synthesis (Figure 4G). The transcription factors SREBP1 and SREBP2 are established regulators of gene expression in lipid synthesis (Espenshade and Hughes, 2007). TCR signals strongly upregulated protein expression of SREBP1/2, including the full-length and processed mature forms. Deficiency of Raptor attenuated both forms of these factors (Figure 4H). In contrast, mRNA encoding SREBP1 and SREBP2 (Srebf1 and Srebf2) was not affected by TCR stimulation regardless of Raptor sufficiency (Figure 4I). Therefore, Raptor promotes the lipogenic program and regulates SREBP functions through post-transcriptional mechanisms.

We further employed GSEA analysis to directly compare global gene expression between WT and Rptor−/− T cells at 8 h of stimulation. Aside from glucose and cholesterol metabolism (Figure S4F), oxidative phosphorylation was also among the most significantly downregulated gene sets in Rptor−/− T cells (Figure S4G). Consistent with the altered gene expression, oxygen consumption rate (OCR) that denoted mitochondrial respiration was reduced in Rptor−/− T cells stimulated with TCR and CD28 (Figure 4J). Collectively, Raptor is required to regulate TCR-induced glycolysis, lipid biosynthesis and oxidative phosphorylation. Given the recent evidence implicating the importance of lipid synthesis and mitochondrial activity in T cell activation (Kidani et al., 2013; Sena et al., 2013), we propose that coordination of these discrete metabolic programs by mTORC1 orchestrates T cell activation and proliferation. Further, the differential kinetics for the induction of metabolic and cell cycle genes at 8 and 24 h (Tables S1 and S2) highlight a Raptor-dependent temporal transition from metabolic reprogramming to cell cycle entry following TCR stimulation.

Raptor is crucial for Th2 cell differentiation

Having shown a central role of Raptor in programming metabolic activities and quiescence exit that differentially impact cell cycle initiation and progression, we next explored the extent to which this pathway was operative for subsequent T cell differentiation. Previous studies have established a requirement of mTORC1 in Th1 and Th17 cell differentiation (Delgoffe et al., 2011; Kurebayashi et al., 2012), but loss of Rheb-dependent mTORC1 activity does not impact Th2 cell development (Delgoffe et al., 2011). However, we found that lack of Raptor markedly decreased IL-4 production under Th2 conditions (Figure 5A), associated with defective Il4 and Il13 mRNA expression (Figure 5B). CFSE labeling showed that CFSElo Rptor−/− cells, representative of those that had entered cell cycle, were impaired to produce IL-4 (Figure 5C). Thus, Raptor function was essential for Th2 cell differentiation in vitro, and this effect likely occurred independently of, or in addition to, its role in cell proliferation.

Figure 5. Raptor deficiency impairs Th2 cell differentiation in vitro and suppresses allergic airway inflammation in vivo.

Figure 5

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(A,B) T cells were cultured under Th2 conditions for 5–6 d, followed by intracellular staining of IL-4 and IFN-γ upon PMA and ionomycin restimulation (A, left), bioplex measurement of IL-4 secretion upon restimulation with α-CD3 for 24 h (A, right), or real-time PCR analysis of Il4 and Il13 mRNA expression upon restimulation with α-CD3 for 5 h (B). (C) Intracellular staining of IL-4 in CFSE-labeled T cells cultured under Th2 conditions. (D) Histopathology of lung in OVA-challenged mice. Left, images of hematoxylin and eosin staining with 2× (upper) or 10× (bottom) of original magnification. Right, histological scores of the lung. (E) Quantification of eosinophils (Eos), neutrophils (Neu), dendritic cells (DC), and T cells (T) in lung and BAL fluid from OVA-challenged mice (n=4–7 mice per group). NS, not significant. (F) Intracellular staining of IL-4 in CFSE-labeled WT CD4+ cells cultured under Th2 conditions in the presence of mock or rapamycin (50 nM) added at different time points. (G) Intracellular staining of IL-4 in CD4+ T cells cultured under Th2 conditions in the presence of mock or rapamycin (50 nM). (H) Intracellular staining of IL-4 in CD4+ T cells cultured under Th2 conditions. Data are representative of 3 (A–C) or 2 (D–H) independent experiments, and error bars represent the SEM. See also Figure S5.

To determine the role of Raptor in Th2 cell responses in vivo, we immunized WT and Rptor−/−mice with OVA and aluminum hydroxide and then challenged them with OVA inhalation. In this model of Th2 cell-dependent allergic airway disease, Raptor deficiency diminished lung inflammation and infiltration of leukocytes including eosinophils into the lung and bronchioalveolar lavage (BAL) fluid (Figures 5D,E). For the few mutant CD4+ T cells that had infiltrated into the lung, they had a markedly reduced ability for IL-4 production (Figure S5A). Furthermore, the BAL fluid from Rptor−/− mice contained markedly reduced levels of IL-4 and Th2-dependent IgE antibody (Figure S5B). These results establish a central role of Raptor in Th2 cell responses in vitro and in vivo.

We next determined the kinetic and signaling requirements for Raptor-dependent cell fate decisions. First, we found that rapamycin treatment at the beginning of the culture profoundly diminished Th2 cell differentiation. This inhibitory effect was less potent, although still evident, when rapamycin was added at later time points (Figure 5F). These results revealed the requirement of a rapamycin-sensitive pathway, likely mTORC1, during the quiescence exit period for shaping Th2 cell differentiation. However, since rapamycin also inhibits mTORC2 activity upon chronic treatment (Delgoffe et al., 2011), an alternative interpretation is that mTORC2 was targeted by rapamycin to influence Th2 cell differentiation, a plausible notion based on the reported roles of mTORC2 in this process (Delgoffe et al., 2011; Lee et al., 2010). To test the contribution of mTORC2, we treated Rictor−/− cells with rapamycin. Whereas loss of Rictor modestly (twofold or less) lowered Th2 cell differentiation (Delgoffe et al., 2011; Lee et al., 2010), rapamycin exerted a marked effect to further diminish IL-4 production from these cells (Figure 5G). Furthermore, loss of both Raptor and Rictor caused very little further reduction in IL-4 as compared with Raptor deficiency alone (Figure 5H). These results illustrate the predominant involvement of mTORC1 over mTORC2 in Th2 cell differentiation.

mTORC1 couples glucose metabolism to cytokine responsiveness

Given a key role of Raptor in mediating T cell glycolysis and lipogenesis, we tested whether the metabolic programs contributed to Th2 cell differentiation. Inhibition of glycolysis with 2-DG had a strong effect to diminish Th2 cell differentiation in a dose-dependent manner, which was evident even when cell proliferation was not affected (Figure 6A). Additionally, 2-DG treatment at the beginning of differentiation was more effective to block Th2 cell differentiation than at later time points of treatment (Figure 6B). In contrast, 25-hydroxycholesterol (25-HC) had a more modest effect on Th2 cell differentiation (Figure S6A), even though it strongly inhibited T cell proliferation. Thus, among the metabolic programs coordinately regulated by Raptor, glucose metabolism appears to play a key role in Th2 cell development.

Figure 6. Raptor links glucose metabolism to cytokine receptor expression and Stat activation for the initiation of Th2 cell differentiation.

Figure 6

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(A) Intracellular staining of IL-4 in CFSE-labeled WT CD4+ cells cultured under Th2 conditions in the presence of mock or 2-DG added at the time of activation. (B) Intracellular staining of IL-4 and IFN-γ in WT CD4+ cells cultured under Th2 conditions in the presence of mock or 2-DG (0.3 mM) added at various times. (C) Expression of IL-4Rα on CD4+ cells cultured under α-CD3-CD28 alone or Th2 conditions for 24 h. (D–F) Flow cytometry of Stat6 phosphorylation (D), IL-2Rα (E), and Stat5 phosphorylation (F) in CD4+ cells cultured under Th2 conditions for 24 h. (G) Expression of IL-4Rα on CD4+ cells cultured under Th2 conditions in the presence of mock, 2-DG or rapamycin for 24 h. (H) Expression of IL-4Rα on CD4+ cells cultured under Th2 conditions in the presence of various doses of glucose. Mean fluorescent intensity (MFI) for the relevant staining is presented above the plots (C–F). Data are representative of 2 (A,B,G,H) or 3 (C–F) independent experiments, and error bars represent the SEM. See also Figure S6.

We further investigated the downstream mechanisms by which mTORC1-dependent metabolism impinges upon Th2 cell differentiation. Development of effector T cells requires the induction of cytokine receptors that in turn confers enhanced responsiveness to cytokines to potentiate T cell differentiation in a feed-forward manner (Zhu et al., 2010). Rptor−/− T cells were impaired to induce IL-4Rα upon stimulation with α-CD3-CD28 or under Th2 cell-polarizing conditions, whereas Rictor−/− cells showed largely normal IL-4Rα induction (Figure 6C). Further, Rptor−/−Rictor−/− T cells showed a similar defect in IL-4Rα induction as Rptor−/− cells (Figure S6B). Consistent with the defective IL-4Rα expression, Rptor−/− T cells were unable to efficiently phosphorylate Stat6 under Th2 cell conditions (Figure 6D). In addition to IL-4, Th2 cell differentiation is also contingent on IL-2 signaling (Zhu et al., 2010). Indeed, Rptor−/− T cells exhibited impaired induction of IL-2Rα (Figure 6E) as well as downstream Stat5 activation (Figure 6F). In contrast, expression of other receptors such as CD122 (IL-2Rβ), CD132 (γc), CD44 and CD69 was largely undisturbed in the mutant cells (Figure S6C). Also, Rptor−/− cells expressed the Th2 factor GATA3 at a lower level than WT cells at 24 h of Th2 cell differentiation (Figure S6D), although this impairment did not persist at later times (data not shown). Therefore, mTORC1 is important for the induction of selective cytokine receptors that likely mediate the enhanced responsiveness to Th2-polarizing cytokines, thereby programming initiation of Th2 cell differentiation.

Are these effects on cytokine receptor expression dependent upon T cell metabolism? To address this, we used selective metabolic inhibitors and measured induction of IL-4Rα. Inhibition of glucose metabolism by 2-DG or of mTOR activity by rapamycin lowered induction of IL-4Rα (Figure 6G). In contrast, 25-HC did not exert strong effects on IL-4Rα expression (Figure S6E). Similarly, rapamycin and 2-DG but not 25-HC inhibited the induction of IL-2Rα (Figures S6E,F). We then explored whether cytokine receptor induction is dependent upon glucose levels in the culture medium. Indeed, lowering glucose levels markedly reduced the expression of IL-4Rα (Figure 6H). We conclude that mTORC1-dependent glucose metabolism contributes to cytokine receptor expression for Th2 cell differentiation.

Raptor integrates TCR and CD28 signals for T cell activation and differentiation

We investigated upstream signals that activate mTORC1-dependent metabolism in Th2 cell differentiation. First, we measured glycolytic activity in α-CD3 activated T cells supplemented with Th2-polarizing cytokines IL-2 and IL-4, but found these cytokines had little effect on glucose metabolism (Figure 7A). In contrast, CD28 co-stimulation markedly potentiated glycolysis (Frauwirth et al., 2002) (Figure 7A), and also enhanced T cell growth, upregulation of nutrient receptors, and lipid synthesis in TCR-stimulated cells (Figure S7A). Second, we determined whether CD28 co-stimulation influenced mTORC1 activity. CD28 had little effects on TCR-induced mTORC1 activation at early time points (Figure S7B), but it potently enhanced mTORC1 after 18 h of stimulation as compared with TCR alone (Figure 7B). Thus, CD28 acts in synergy with TCR to potentiate T cell metabolism and sustain mTORC1 activation.

Figure 7. Raptor but not Rheb is essential for TCR and CD28-induced sustained mTORC1 activation and physiological effects.

Figure 7

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(A) Glycolytic activity of WT CD4+ T cells stimulated with α-CD3 or α-CD3-CD28 in the presence of IL-2, IL-4 or both for 24 h. (B) Phosphorylation of S6K1, S6 and 4E–BP1 in WT CD4+ T cells stimulated with α-CD3 or α-CD3-CD28. (C) BrdU staining of CD4+ T cells stimulated with α-CD3-CD28 for 22 h, followed by pulsing with BrdU for 2 h. (D) Intracellular staining of IL-4 and IFN-γ in CD4+ T cells cultured under Th2 conditions. (E) Phosphorylation of 4E-BP1, S6K1 and S6 in CD4+ T cells stimulated with α-CD3 or α-CD3-CD28. (F) Phosphorylation of S6K1, 4E-BP1 and AKT Ser473 in CD4+ T cells stimulated with α-CD3 or α-CD3-CD28. Data are representative of 1 (A) or 2 (B–F) independent experiments, and error bars represent the SEM. See also Figure S7.

To further explore the signaling mechanisms, we determined whether Rheb is involved by directly comparing the effects of Rheb and Raptor deficiencies on T cell activation, Th2 cell differentiation and mTORC1 activity. As compared with the profound defects in T cell proliferation and Th2 cell differentiation in Rptor−/− cells, Rheb−/− cells exhibited much milder defects in cell cycle entry (Figure 7C) and proliferation (Figures S7C,D), and normal Th2 cell differentiation (Delgoffe et al., 2011) (Figure 7D). Further, whereas Raptor deficiency abrogated phosphorylation of 4EBP1, S6K1 and S6 at all the time points, loss of Rheb blocked mTORC1 activation only at early time of TCR stimulation, but the activity largely recovered at 18 h (Figure 7E). Thus, Rheb is not essential for sustained mTORC1 activation induced by TCR and co-stimulation. We next investigated whether mTORC2 activity is involved in this process. Loss of Rictor ablated AKT Ser473 phosphorylation but had no effect on mTORC1 activation (Figure 7F), consistent with previous observations (Lee et al., 2010). Altogether, Raptormediated mTORC1 activation integrates TCR and CD28 co-stimulation for T cell responses (Figure S7E), and this process can be regulated independently of Rheb or mTORC2.

DISCUSSION

Antigen-induced clonal expansion is a hallmark of adaptive immunity, but little is known how this process is initiated, namely how naïve T cells respond to antigens by transitioning from programmed quiescence into active cycling. Here we describe a pathway in which Raptor-dependent mTORC1 activation and metabolic reprogramming drive the exit of T cells from quiescence, and this in turn coordinates T cell activation and fate decisions. Mechanistically, Raptor-mTORC1 orchestrates cell metabolism especially the glycolytic and lipogenic programs to facilitate the entry into an active cell cycle. Further, this pathway is intimately linked to fate decisions, as deficiency of Raptor or blocking glucose metabolism prevents the generation of Th2 cells. Direct comparisons of phenotypic alterations in T cells lacking Raptor with those deficient in Rheb or Rictor highlight an unexpected, predominant contribution of Raptor-mTORC1 to T cell activation and Th2 cell differentiation. We propose that Raptor-dependent metabolic reprogramming and quiescence exit are a fundamental determinant that underlies specific proliferative and differentiative responses in adaptive immunity.

Our results highlight that quiescence exit is an intermediate state connecting early TCR signaling (e.g. NF-κB and MAPK activation) and cell cycle entry, marked by mTORC1-dependent extensive metabolic reprogramming. However, mTORC1 is less crucial for continuous proliferation. The simplest interpretation is that cell cycle entry of naïve T cells has a stringent requirement of metabolic activities. Once T cells have entered active cycling, the metabolites and intermediates synthesized at the time of exiting quiescence may allow them to engage compensatory pathways when mTORC1 is inhibited. Despite this, the delay in quiescence exit in Rptor−/− cells is functionally important as this essentially cripples adaptive immunity. These studies have established a critical cellular context whereby mTORC1-dependent metabolism promotes cell proliferation. Further, mTORC1 activated within the first 24 h makes a profound contribution to Th2 cell differentiation, highlighting mTORC1-mediated quiescence exit as a central mechanism in T cell activation and fate decisions.

For T cell differentiation, much of the recent emphasis has been placed on cytokine receptor signaling. In contrast, the mechanisms by which co-stimulation shapes T cell fates are poorly understood, despite earlier studies implicating their functional significance (Corry et al., 1994). Moreover, relatively little is known about the initiation phase of T cell differentiation (Yamane and Paul, 2013). Here we show that Raptor-mTORC1 integrates TCR and CD28 signals and links them to T cell metabolism and cytokine responsiveness as an important mechanism to program Th2 cell differentiation. Deletion of Raptor or blocking glucose metabolism impairs proper differentiation into Th2 effector cells, associated with defective cytokine receptor expression and Stat activation. As for the stringent requirement of this pathway in Th2 cell differentiation , it is important to note that Th2 cell differentiation is metabolically demanding in that Th2 cells have the highest rate of glycolysis among all effector T cells examined (Michalek et al., 2011; Shi et al., 2011). Furthermore, effector cytokine expression, especially IL-4 from Th2 cells, is cell cycle-dependent (Bird et al., 1998). Whereas our finding certainly does not exclude the contribution of mTORC1-dependent proliferation to Th2 cell differentiation, Raptor deficiency impairs multiple processes required for Th2 cell generation at 24 h of activation, before the first cell division. Additionally, Rptor−/− cells that have undergone division (CFSElo) cells are capable of continuous division but not for IL-4 production. Furthermore, 2-DG inhibits Th2 cell differentiation, even at doses without affecting cell proliferation. Altogether, we propose that Raptor-mediated metabolic reprogramming is an underlying mechanism to couple proliferation and Th2 cell differentiation, thereby placing metabolic fitness as a key upstream determinant to coordinate both processes (Figure S7E).

In contrast, mTORC1-dependent Th2 cell differentiation is independent of Rheb, in agreement with a previous report (Delgoffe et al., 2011). We also noted that deletion of Raptor is more detrimental for embryonic and brain development than Rheb deficiency in (Cloetta et al., 2013; Goorden et al., 2011; Guertin et al., 2006; Zou et al., 2011). These studies suggest the existence of functional redundancy in the GTPases or the presence of alternative pathways for mTORC1 activation. Although mTORC2 contributes to Th2 cell differentiation (Delgoffe et al., 2011; Lee et al., 2010), we show that it plays a more modest role than Raptor. Further, loss of Rictor has little effect on the activation of mTORC1 or IL-4Rα expression in T cells. Moreover, AKT activity is required for Th1 but not Th2 cell differentiation (Kane et al., 2001). Altogether, these results indicate the presence of novel upstream inputs independent of Rheb, mTORC2 or AKT for the activation of mTORC1 and Th2 cell differentiation. We speculate this could involve the PI3K-AKT-independent pathway for mTORC1 activation in activated CD8+ T cells that is mediated by the kinase PDK1 (Finlay et al., 2012; Macintyre et al., 2011) and/or amino acid uptake (Sinclair et al., 2013). Future work is required to identify the nature of such signals in CD4+ T cell responses.

Emerging evidence points to an important role for metabolic control of T cell functions and fates orchestrated by transcription factors (Wang and Green, 2012). However, there is little genetic evidence linking mTOR to T cell metabolism. Here we have identified a central role of Raptor-mTORC1 in coordinating multiple metabolic pathways, including glycolysis, lipid synthesis and oxidative phosphorylation. This is accompanied by dynamic transcriptional induction of metabolic enzymes and post-translational regulation of key transcription factors including c-Myc and SREBPs. A similar requirement for mTORC1 in glycolysis and lipogenesis has been shown in growth factor-independent proliferation of mouse embryonic fibroblasts (Duvel et al., 2010). However, the effector mechanisms downstream of mTORC1 are distinct, which could reflect physiologically programmed quiescence and signal-dependent exit that are unique to lymphocytes. Further, our recent studies have established a crucial role of mTORC1-dependent lipogenic pathway in establishing regulatory T cell function (Zeng et al., 2013). These findings collectively highlight cell context-specific regulation and function of mTORC1-dependent metabolic network.

In summary, our study has identified a key role of mTORC1-mediated metabolic reprogramming for mediating T cell exit from quiescence that in turn impacts T cell proliferation and fate decisions. Although metabolic upregulation is generally thought to support the high rate of cell proliferation, our results suggest that mTORC1-dependent metabolism is more important for the exit from quiescence and initiation of the cell cycle, i.e. the ‘competence’ to enter this high rate of expansion, rather than maintaining the high rate of active proliferation. Moreover, the initial metabolic reprogramming has a crucial role in imprinting the ensuing fate decisions. We propose that the unique metabolic requirement of quiescence exit defines one of the major cellular events connecting early signals from TCR and co-stimulation and subsequent proliferative and differentiative responses in adaptive immunity.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6, CD45.1+, OT-II, and Rag1−/− mice were purchased from the Jackson Laboratory. Rptorfl, Rictorfl and CD4-Cre mice have been described (Yang et al., 2011; Zeng et al., 2013). The generation of Rheb floxed mice will be described elsewhere. All mice were kept in specific pathogen-free conditions in Animal Resource Center at St. Jude. Animal protocols were approved by Institutional Animal Care and Use Committee of St. Jude.

Cell purification and cultures

Sorted naïve T cells (CD4+CD62LhiCD44loCD25−) were used for in vitro cultures in Click’s medium supplemented with β -mercaptoethanol, 10% FBS and 1% penicillin-streptomycin. For Th2 cell differentiation, naïve T cells were activated with α -CD3 (2C11; 2 µg/ml), α -CD28 (37.51; 2 µg/ml), human IL-2 (100 U/ml), IL-4 (10 ng/ml), α-IFN-γ (10 µg/ml) and irradiated splenocytes as antigen-presenting cells. Live cells were collected by Ficoll purification after 5–6 d of differentiation, and restimulated with PMA and ionomycin for intracellular staining, or with anti-CD3 for mRNA and bioplex analysis. To measure cytokine receptors and transcription factors at early time points (24 h), naïve T cells (CD45.2+) were stimulated as above except in the presence of irradiated splenocytes (CD45.1+) to unequivocally identify T cells. To alter glucose concentrations, T cells were cultured under Th2 conditions in glucose-free DMEM medium, 10% dialyzed FBS and various doses of glucose. To obtain protein extracts used for immunoblots, T cells were incubated with biotinylated α -CD3-CD28 followed by crosslinking streptavidin for short-term stimulation, or stimulated with plate-bound α-CD3-CD28 for long-term stimulation. Retroviral transduction of T cells was performed as described previously (Liu et al., 2010).

Metabolic assays

CD4+ T cells were stimulated with plate-bound α-CD3-CD28 for 24 h, and glycolytic flux was measured by detritiation of [3-3H]-glucose, as previously described (Shi et al., 2011). De novo lipid synthesis was determined as described (Duvel et al., 2010; Zeng et al., 2013). Briefly, for the final 4 h of culture, [1-14C]acetic acid (Perkin Elmer) was added to the cells. Cells were lysed in 0.5% Triton X-100, and the lipid fraction was extracted by addition of chloroform and methanol (2:1 v/v) with vortexing, followed by addition of water with vortexing. After centrifugation, the lipid-containing phase was obtained and 14C incorporation was measured using a Beckman LS6500 scintillation counter. The OCR rate was measured using the Seahorse XF24-3 extracellular flux analyzer per manufacturer’s instructions (Seahorse Bioscience).

Statistical analysis

P values were calculated using Student’s t-test. P values of less than 0.05 were considered significant.

Supplementary Material

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HIGHLIGHTS.

  1. mTORC1-dependent metabolic reprogramming drives exit of T cells from quiescence

  2. mTORC1 coordinates T cell glycolysis, lipogenesis and oxidative phosphorylation

  3. Raptor links glucose metabolism to cytokine responsiveness and Th2 cell generation

  4. Raptor integrates TCR and CD28 co-stimulation signals

ACKNOWLEDGEMENTS

The authors acknowledge the D. Green laboratory for help with the Seahorse analyzer, R. Cross, G. Lennon and P. Ingle for cell sorting, and Y. Wang for editing. This work was supported by US National Institutes of Health (R01 AI101407, R01 NS064599 and R21 AI094089) and American Cancer Society (RSG-13-248-01-LIB) (H.C.), and by a postdoctoral fellowship from the Arthritis Foundation (K.Y.).

Footnotes

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ACCESSION NUMBERS

The microarray data are available in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds) under the accession number GSE51668.

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures, two tables, Supplemental Experimental Procedures, and Supplemental References.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS542075-supplement-01.pdf (3.4MB, pdf)
02
NIHMS542075-supplement-02.xlsx (135.7KB, xlsx)
03
NIHMS542075-supplement-03.xlsx (39.1KB, xlsx)

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