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A Role for Mammalian Target of Rapamycin in Regulating T Cell Activation versus Anergy¹

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University Medical Institutions, Baltimore, MD 21231

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Whether TCR engagement leads to activation or tolerance is determined by the concomitant delivery of multiple accessory signals, cytokines, and environmental cues. In this study, we demonstrate that the mammalian target of rapamycin (mTOR) integrates these signals and determines the outcome of TCR engagement with regard to activation or anergy. In vitro, Ag recognition in the setting of mTOR activation leads to full immune responses, whereas recognition in the setting of mTOR inhibition results in anergy. Full T cell activation is associated with an increase in the phosphorylation of the downstream mTOR target S6 kinase 1 at Thr⁴²¹/Ser⁴²⁴ and an increase in the mTOR-dependent cell surface expression of transferrin receptor (CD71). Alternatively, the induction of anergy results in markedly less S6 kinase 1 Thr⁴²¹/Ser⁴²⁴ phosphorylation and CD71 surface expression. Likewise, the reversal of anergy is associated not with proliferation, but rather the specific activation of mTOR. Importantly, T cells engineered to express a rapamycin-resistant mTOR construct are resistant to anergy induction caused by rapamycin. In vivo, mTOR inhibition promotes T cell anergy under conditions that would normally induce priming. Furthermore, by examining CD71 surface expression, we are able to distinguish and differentially isolate anergic and activated T cells in vivo. Overall, our data suggest that by integrating environmental cues, mTOR plays a central role in determining the outcome of Ag recognition.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In a two-signal model of immune activation, the ultimate outcome of an immune response is not dictated by the Ag itself, but by the context in which the Ag is encountered. Signal 1 refers to TCR engagement that heralds recognition, whereas signal 2 refers to costimulation and is necessary for the induction of full T cell activation (1). In in vitro models, signal 1 alone leads to anergy and signal 2 can be delivered by the cross-linking of CD28. In vivo, however, signal 2 involves multiple inputs, both activating and inhibitory, from multiple accessory molecules and cytokines. Consequently, when a T cell encounters Ags, it must also sense and integrate environmental cues to respond appropriately.

The mammalian target of rapamycin (mTOR)³ is an evolutionarily conserved 289-kDa serine/threonine protein kinase that is inhibited by rapamycin (2). In yeast and mammalian cells, TOR integrates environmental cues in terms of nutrients, energy, and growth factors, and directs cell growth and proliferation. Insulin and other growth factors activate mTOR in a PI3KAKT-dependent fashion. AKT in turn phosphorylates and hence inactivates tuberous sclerosis complex 2, a potent inhibitor of mTOR activity (3). Growth factors also have the ability to activate mTOR through an AKT-independent pathway that involves the activation of phospholipase D and the generation of phosphatidic acid (4). Alternatively, mTOR is inhibited when there is a lack of energy stores. Under these conditions, an increase in the ratio of AMP:ATP leads to the activation of AMP kinase that in turn inhibits mTOR by promoting tuberous sclerosis complex 2 activity (3). Although the list of downstream targets of mTOR is continuously expanding, one of the most studied readouts of mTOR function is the phosphorylation of S6 kinase-1 (S6K-1), which is a translational regulator (5).

Previously, our group and others demonstrated that the mTOR inhibitor rapamycin promoted T cell anergy even in the presence of costimulation (6, 7). Initially, this was thought to be due to the ability of rapamycin to block cell cycle progression. Indeed, multiple investigators have correlated anergy with blockade in the cyclin-dependent kinase cascade (8, 9, 10, 11). However, it is becoming increasingly clear that the induction of anergy can be disassociated from cell cycle arrest (12, 13). In this study, we provide further evidence that TCR engagement in the absence of cell cycle progression does not necessarily lead to anergy and cell proliferation does not necessarily reverse anergy. Furthermore, we test the hypothesis that mTOR plays a role in dictating the outcome of TCR engagement. Just as mTOR plays a critical role in integrating environmental cues for regulating growth and differentiation, we propose that mTOR plays a central role in integrating the environmental signals that determine whether Ag recognition will lead to activation or tolerance.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Reagents and Abs

Rapamycin (a gift from S. Sehgal, Wyeth-Ayerst, Princeton, NJ) was dissolved in ethanol. For in vivo injection, rapamycin was dissolved in carboxymethylcellulose (Sigma-Aldrich). Sanglifehrin A (SFA; a gift from R. Sedrani, Novartis, Basel, Switzerland) was dissolved in DMSO. Pigeon cytochrome c (PCC) peptide (81–104; FAGIKKKAERADLIAYLKQATAK) for injection was a gift from H. Yu (Johns Hopkins University, Baltimore, MD), and PCC peptide used in in vitro stimulation was purchased from Sigma-Aldrich. CFSE was obtained from Molecular Probes. Total S6K-1 and phospho-S6K-1 (Thr⁴²¹/Ser⁴²⁴) were obtained from Cell Signaling Technology. Total AKT and phospho-AKT (Ser⁴⁷³) were from Upstate Biotechnology. p27^Kip1 and cyclin D3 Abs were purchased from Santa Cruz Biotechnology. Actin Ab was obtained from Sigma-Aldrich. Anti-rabbit IgG HRP-linked Ab was from Amersham Biosciences. Conjugated Abs for CD71 and V3 were from eBioscience and BD Pharmingen.

Cell culture and mice

A.E7, a CD4⁺ Th1 clone specific for PCC peptide 81–104, was maintained as previously described (6). Jurkat cells were obtained from American Type Culture Collection and maintained in RPMI 1640 with 10% FBS (Invitrogen Life Technologies). The 293T cells were purchased from American Type Culture Collection and cultured in DMEM with 10% FBS (Invitrogen Life Technologies). B10.A and 5C.C7 mice were purchased from Taconic Farms. The 5C.C7 mice are TCR transgenic (specific for the I-E^k-restricted PCC 81–104 peptide) on a Rag2^–/– B10.A background. The studies have been reviewed and approved by the animal review committee at Johns Hopkins Medical Institutions.

Constructs

Bicistronic lentivirus vector EF.CMV.GFP (a gift from X. Yu, Johns Hopkins University, Baltimore, MD) in which the gene of interest is driven by a human EF1- promoter, and GFP by a CMV promoter. Wild-type mTOR (WT-mTOR) and rapamycin-resistant mTOR (RR-mTOR) were cut from pCDNA3-WT-mTOR and RR-mTOR vectors (a gift from J. Chen, University of Illinois, Urbana, IL), and subcloned into the bicistronic lentiviral vector to make EF.WT-mTOR-CMV.GFP and EF.RR-mTOR-CMV.GFP constructs.

In vitro anergy model

A total of 5 x 10⁶ A.E7 cells was stimulated with plate-bound anti-CD3 (1 µg/ml, 2C11; BD Pharmingen) to induce anergy, or stimulated with plate-bound anti-CD3 plus soluble anti-CD28 (1/1000 of ascitic fluid containing anti-CD28 mAb 37.51; a gift from J. Allison, Sloan Kettering, New York, NY) to be activated. After 16 h, the cells were washed three times with PBS to remove Abs and rested in medium for 5–7 days. Upon rechallenge, 5 x 10⁴ A.E7 cells were stimulated with 5 x 10⁵ irradiated B10.A splenocytes (served as APCs) and PCC peptide (0–0.5 µM); proliferation was measured by thymidine incorporation after 48 h. To determine IL-2 production upon rechallenge, the treated cells were stimulated with plate-bound anti-CD3 (1 µg/ml) plus soluble anti-CD28 (1/1000), supernatant was collected after 24 h, and IL-2 was measured by ELISA (eBioscience). To study anergy reversal, A.E7 cells were anergized with 1 µg/ml plate-bound anti-CD3 for 16 h, washed, and rested in medium supplemented with either 100 ng/ml IL-2 (BioSource International) or 10 ng/ml IL-7 (BioSource International) for 5–7 days. Then the cells were washed three times with PBS to remove residual cytokines and rechallenged, as described above.

Transfection of Jurkat cells

To transfect Jurkat cells, 6 µg of lentiviral constructs was added to 12 x 10⁶ cells in 300 µl of Jurkat medium without antibiotics and incubated for 15 min at room temperature, and the cells were then electroporated at 300 V, 24 , and 800 µF using BTX EMC600 electroporator.

Infection of 5C.C7 T cells

To make recombinant lentivirus, 293T cells (10 x 10⁶) were seeded onto 150-mm dishes, cultured for 16 h, and transiently transfected with 9 µg of lentivirus construct, 12 µg of pCMVR8.91, plus 3 µg of pMDG plasmids using Lipofetamine 2000 (Invitrogen Life Technologies). The virus supernatants were collected at 24, 48, and 72 h, and concentrated using filtration columns (Centricon Plus-70, molecular mass cutoff 100 kDa; Millipore). Viral titer was determined by transduction of 293T cells (2 x 10⁵ cells/well in 6-well plates) with serially diluted virus supernatant plus 8 µg/ml polybrene (Sigma-Aldrich), and the percentage of GFP⁺ cells was assessed 3 days later by FACS. To transduce 5C.C7 cells, spleens and lymph nodes were harvested from 5C.C7 mice and activated by PCC peptide (5 µM). Forty-eight hours later, 3 x 10⁶ activated 5C.C7 cells, 8 µg/ml polybrene, and recombinant lentivirus (at multiplicity of infection of 1) were placed in 12-well plates, centrifuged for 3 h at 1800 x g, 32°C, and cultured for another 16 h. Cells were then washed and rested in fresh A.E7 medium supplemented with 1 ng/ml IL-2 and 10 ng/ml IL-7 for 5 days before being sorted by BD FACSAria.

In vivo peptide-induced anergy model

Splenocytes and lymphocytes from 5C.C7 TCR transgenic Rag2^–/– mice (CD4⁺V3⁺) were labeled with CFSE, and 4–5 x 10⁶ cells per mouse were adoptively transferred into syngeneic B10.A hosts by i.v. injection (8–10 x 10⁶ cells per mouse for the naive condition to increase recovery, because these cells do not expand). On the second day, the mice received i.v. either 150 µg of PCC peptide (81–104) or HBSS (vehicle). For rapamycin-treated mice, rapamycin was prepared at 2 mg/ml in carboxymethylcellulose, and injected i.p. at a dose of 1.5 mg/kg beginning on the day of adoptive transfer, and continuing daily till the end of the experiment. On day 4, spleens were harvested, and CD4⁺ T cells were isolated by negative selection using MACS CD4⁺ T cell isolation kit and MACS LS separation columns (Miltenyi Biotec). Next, CFSE low V3⁺ clonotypic cells were sorted by BD FACSAria, and 2 x 10⁴ sorted cells were rechallenged with 2 x 10⁵ syngeneic irradiated B10.A splenocytes and 0.5–5 µM PCC peptide. Supernatant was collected 48 h later, and IL-2 was analyzed by ELISA.

Immunoblot analysis

To analyze mTOR activity during anergy induction, A.E7 cells were stimulated with anti-CD3 (1 µg/ml, plate bound) alone, anti-CD3 plus anti-CD28 (1/1000, soluble), anti-CD3 plus anti-CD28 plus SFA (1 µM), or rapamycin (1 µM) for 16 h in A.E7 medium. To analyze mTOR activity during anergy reversal, A.E7 cells were anergized with 1 µg/ml plate-bound anti-CD3 for 16 h, washed, and rested in A.E7 medium alone, or A.E7 medium supplemented with either 100 ng/ml IL-2 or 10 ng/ml IL-7 for 4 days. Next, the cells were washed three times with PBS and cultured for 4 h in the same previous condition in the absence of serum. To compare the effect of IL-2 and IL-7 on primary T cells, splenocytes from 5C.C7 mice were harvested and activated with 5 µM PCC peptide for 48 h, followed by a 5-day expansion in medium supplemented with 1 ng/ml IL-2. These preactivated primary 5C.C7 T cells were then washed with PBS three times and cultured with 100 ng/ml IL-2 or 10 ng/ml IL-7. To make cell lysate, the treated cells were harvested and resuspended in ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 0.5% Nonidet P-40, 5 mM EDTA, 50 mM NaCl, and 1x protease inhibitor mixture (Roche). After a 15-min incubation, the cells were spun and supernatant was collected for evaluation of protein concentration by bicinchoninic acid protein assay kit (Pierce). Equal amount of extracts was loaded on 10% Bis-Tris minigel (Invitrogen Life Technologies), separated by SDS-PAGE, and followed by transfer to nitrocellulose membrane (Invitrogen Life Technologies). Western blot analysis was performed using Abs listed above and corresponding secondary Abs.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TCR engagement in the absence of mTOR activation induces T cell anergy

It is clear that anergic T cells fail to undergo cell cycle progression; however, whether this is a cause or consequence of anergy remains controversial. Although it has been proposed that TCR engagement in the absence of cell cycle progression causes anergy, recently, we have demonstrated that the novel cyclophilin-binding compound SFA could block cell cycle progression in G₁ without inducing anergy (13). In contrast, we and others have shown that inhibiting cell cycle progression with rapamycin (which also blocks cell cycle progression in G₁) does induce anergy (6, 7). Thus, we wanted to exploit the differences between these two inhibitors and try to dissect the biochemical mechanisms contributing to anergy induction. As seen in Fig. 1A, stimulation of the PCC-specific Th1 T cell clone A.E7 with anti-CD3 and anti-CD28 (signal 1 + 2) led to proliferation. Both rapamycin and SFA equally inhibited proliferation. However, as seen in Fig. 1B, blocking cell cycle progression with rapamycin (during the initial stimulation or induction phase) led to anergy such that the cells failed to produce IL-2 upon full rechallenge 7 days later. In contrast, inhibition of cell cycle progression with SFA did not result in anergy because these cells produced copious amounts of IL-2 upon rechallenge.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. TCR engagement in the absence of mTOR activation induces T cell anergy. A, Both SFA and rapamycin block T cell proliferation. CFSE-labeled A.E7 cells were stimulated with anti-CD3 plus anti-CD28 (1 + 2), anti-CD3 plus anti-CD28 plus SFA (1 + 2 + SFA), or anti-CD3 plus anti-CD28 plus rapamycin (1 + 2 + R). After 48 h, cell division was analyzed by CFSE dilution. B, Rapamycin, but not SFA, induces anergy. A.E7 cells were stimulated 16 h with anti-CD3 alone (1+), anti-CD3 plus anti-CD28 (1 + 2), anti-CD3 plus anti-CD28 plus SFA (1 + 2 + SFA), or anti-CD3 plus anti-CD28 plus rapamycin (1 + 2 + R). Abs and drugs were then removed, and the cells were rested for 5–7 days. Next, the cells were rechallenged with anti-CD3 plus anti-CD28, and evaluated for IL-2. C, SFA and rapamycin arrest cell cycle progression with different mechanisms. A.E7 cells were left untreated (M) or stimulated 16 h with anti-CD3 (1+), anti-CD3 plus anti-CD28 (1 + 2), anti-CD3 plus anti-CD28 plus SFA (1 + 2 + SFA), or anti-CD3 plus anti-CD28 plus rapamycin (1 + 2 + R). The expression of p27kip1 and cyclin D3 was analyzed by Western blot. The blot was then stripped and reblotted for -actin as a loading control. D, Rapamycin, but not SFA, inhibits mTOR activity. A.E7 cells were stimulated for 16 h with mock (M), anti-CD3 alone (1+), anti-CD3 plus anti-CD28 (1 + 2), anti-CD3 plus anti-CD28 plus SFA (1 + 2 + SFA), or anti-CD3 plus anti-CD28 plus rapamycin (1 + 2 + R). Phospho-S6K-1 was analyzed by Western blot. The blot was then stripped and reblotted for total S6K-1 as a loading control. Data are representative of three independent experiments.

The expression of the cyclins is regulated by the PI3K-related kinase family member mTOR (15). mTOR has multiple downstream targets. For example, the activation of mTOR is typically measured by the phosphorylation of one of its downstream substrates S6K-1 (5, 16, 17, 18, 19). As shown in Fig. 1D, and consistent with our previous findings, rapamycin, but not SFA, inhibited mTOR activity, as determined by the phosphorylation of S6K-1 at Thr⁴²¹/Ser⁴²⁴. There is a basal level of S6K-1 phosphorylation in mock-stimulated cells. TCR stimulation alone induced a modest increase in phospho-S6K-1 levels. In contrast, TCR stimulation in conjunction with costimulation led to a significant up-regulation of phopho-S6K-1, suggesting enhanced mTOR activity. This enhancement was not affected by SFA, but completely inhibited by rapamycin. Overall, these sets of experiments suggest that the induction of anergy might be best correlated with the inhibition of mTOR rather than actual cell cycle progression.

Activation of mTOR reverses T cell anergy

To pursue the potential association between mTOR inhibition and anergy, we next examined mTOR activity during anergy reversal. T cell anergy is not a terminally differentiated state and can be reversed by IL-2. Boussiotis et al. (20) have proposed that signals from common -chain ILs regulate T cell activation vs anergy. IL-7R shares the common -chain with the IL-2R and has been implicated in regulating cell size and glucose metabolism in a PI3K-mTOR-dependent manner (21). Thus, we wanted to determine whether IL-7 could reverse anergy.

CFSE-labeled A.E7 cells were treated by anti-CD3 alone overnight to induce anergy (Fig. 2A). Next, the cells were rested in medium alone, or medium supplemented with IL-2 or IL-7, and proliferation was analyzed by CFSE dilution 4 days later. The anergic cells rested in medium alone did not proliferate (Fig. 2B). In contrast, the anergic cells in either IL-2 or IL-7 proliferated vigorously. Thus, with regard to the ability to promote proliferation, both IL-2 and IL-7 were equally effective. Next, we compared the ability of IL-2 and IL-7 to reverse anergy (Fig. 2, C and D). The anergic cells that were rested in medium alone failed to produce IL-2 or proliferate upon rechallenge; they remained anergic. In contrast, anergy was reversed when the cells were rested in IL-2 because upon rechallenge they produced IL-2 and proliferated. Interestingly, although the cells rested in IL-7 proliferated to the same extent as the cells with IL-2, these cells were still anergic when they were rechallenged.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Proliferation does not necessarily reverse T cell anergy. A, Experimental scheme. B, Anergic cells proliferate in both IL-2 and IL-7. CFSE-labeled A.E7 cells were anergized by plate-bound anti-CD3, and rested in medium alone (1+) or medium supplemented with either IL-2 (1 + IL-2) or IL-7 (1 + IL-7). Cell division was analyzed by CFSE dilution. A.E7 cells that were mock stimulated and rested in medium were used as a control (M). C and D, IL-2, but not IL-7, reverses anergy. A.E7 cells were treated as in A. After a 7-day rest, the cells were extremely washed with PBS to remove residual cytokines, followed by rechallenge. IL-2 production (C) and proliferation (D) were analyzed. Data are representative of three independent experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. A, mTOR activity is associated with anergy reversal. A.E7 cells were anergized by plate-bound anti-CD3 and rested in medium alone (1+) or medium supplemented with either IL-2 (1 + IL-2), IL-7 (1 + IL-7), or IL-2 plus rapamycin (1 + IL-2 + R). After 96 h, mTOR activity was analyzed by phosphorylation of S6K-1. The blot was then stripped and reblotted for total S6K-1 as a loading control. B, Both IL-2 and IL-7 activate AKT. Preactivated 5C.C7 T cells were left untreated (M) or treated for 30 min with IL-2 or IL-7 at the same doses used in anergy reversal (Fig. 2); phosphorylation of AKT Ser473 was assessed. The blot was then stripped and reprobed for total AKT. C, IL-2, but not IL-7, phosphorylates S6K-1 at Thr421/Ser424. Preactivated 5C.C7 T cells were left untreated (M) or treated for 24 h with IL-2 or IL-7 at the same doses as in B; phosphorylation of S6K-1 Thr421/Ser424 was assessed. The blot was then stripped and reprobed for total S6K-1. Data are representative of two independent experiments.

Thus, whereas IL-7 promotes the phosphorylation of AKT (Fig. 3B) and can induce the phosphorylation of S6K-1 at Thr³⁸⁹ (data not shown) (24), unlike IL-2, IL-7 does not promote mTOR-induced phospho-S6K-1 at Thr⁴²¹/Ser⁴²⁴, which is associated with the reversal of anergy.

Transferrin receptor expression correlates with mTOR activity in T cells

mTOR is known to control a wide range of growth- and proliferation-related cellular events. It has been reported recently that the loss of cell surface transporters for glucose, amino acids, low-density lipoproteins, and iron transferrin resulting from growth factor withdrawal occurs in an mTOR-dependent fashion (25). Among these transporters, the iron transferrin receptor CD71 has been shown to be up-regulated on activated T cells (26). As such, CD71 represents a potential cell surface marker for mTOR activation in T cells, and thus a means of distinguishing activated from anergic T cells. We undertook a series of experiments to examine the cell surface expression of CD71 during anergy induction and full activation.

We first analyzed the cell surface expression profile of CD71 during anergy induction (Fig. 4A). A.E7 cells were stimulated with anti-CD3 and anti-CD3 plus anti-CD28 in the absence or presence of rapamycin, and cell surface expression of CD71 was analyzed by FACS. Although the cell surface expression of CD71 is clearly detectable on the cells undergoing anergy induction (anti-CD3 alone), when the cells were stimulated with anti-CD3 plus anti-CD28 the cell surface expression of CD71 was markedly increased. That is, the cell surface expression levels of CD71 could distinguish cells stimulated with signal 1 alone vs signals 1 + 2. The cell surface expression of CD71 is mTOR dependent, as demonstrated by the fact that treatment with rapamycin inhibits its expression. Thus, the relative cell surface expression of CD71 can distinguish T cells in which TCR engagement occurs in the context of costimulation vs those in which Ag recognition occurs in the absence of costimulation.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. CD71 surface expression correlates with mTOR activity in T cells. A, The cell surface expression of CD71 increases upon activation. A.E7 cells were stimulated with anti-CD3 alone (1+), anti-CD3 plus anti-CD28 (1 + 2), or anti-CD3 plus anti-CD28 plus rapamycin (1 + 2 + R), and the cell surface expression of CD71 was analyzed 16 h later by FACS. B and C, The surface expression of CD71 increases when anergy is reversed. Anergic A.E7 cells were rested in medium alone (An), medium with IL-2 (An + IL-2), or medium with IL-2 plus rapamcyin (An + IL-2 + R), and CD71 surface expression was analyzed 24 h later (B). Alternatively, anergic A.E7 cells were rested in medium alone (An), medium with IL-2 (An + IL-2), or medium with IL-7 (An + IL-7) for 7 days, and CD71 surface expression was examined (C). Data are representative of three independent experiments.

Rapamycin promotes T cell anergy by inhibiting mTOR

Thus far, we have been able to demonstrate an association between mTOR activity leading to the phosphorylation of S6K-1 at Thr⁴²¹/Ser⁴²⁴ and full T cell activation. We next wanted to directly test the ability of mTOR activation to prevent anergy induction. Consistent with the role of mTOR in promoting full T cell activation is the observation that inhibiting mTOR with rapamycin promotes T cell anergy even in the presence of costimulation. To prove that rapamycin promotes anergy by inhibiting mTOR, we transfected T cells with a RR-mTOR mutant.

Rapamycin associates with mTOR through the FKBP12-rapamycin binding domain. Ser²⁰³⁵ within this domain is critical for the interaction, and mutations at this site eradicate the ability of rapamycin to bind mTOR such that the mutant mTOR is resistant to rapamycin-induced inhibition (27). Jurkat cells were transfected with an empty lentivirus vector construct, WT-mTOR, or RR-mTOR. As demonstrated in Fig. 5A, in the absence of rapamycin, the constitutive mTOR activity in Jurkat cells (which is a lymphoma cell line) was equivalent in all the transfected cells (top row). In contrast, rapamycin at a concentration of 30 nM inhibited phosphorylation of S6K-1 in the Jurkat cells transfected with empty lentivirus vector or WT-mTOR (middle row). In contrast, even in the presence of 100 nM rapamycin, phospho-S6K-1 could still be detected in the cells expressing RR-mTOR (bottom row). If the ability of rapamycin to promote T cell anergy was in fact due to its ability to inhibit mTOR, then we would expect that T cells expressing the RR-mTOR would be resistant to rapamycin-induced anergy. To test this hypothesis, T cells from 5C.C7 TCR transgenic Rag2^–/– mice were infected with WT- or RR-mTOR recombinant lentivirus, and stimulated with anti-CD3 plus anti-CD28 plus rapamycin at 0, 30, and 100 nM. After a 5-day rest, the infected cells were sorted and rechallenged with APCs plus PCC peptide. As seen in Fig. 5, B and C, rapamycin promoted anergy in the WT-mTOR-expressing cells in a dose-dependent fashion such that upon rechallenge (in the absence of any rapamycin), these cells proliferated less and produced less IL-2. Alternatively, the cells that were transfected with the RR-mTOR construct proliferated and produced IL-2 equivalently to their nonrapamycin-treated counterparts. These data indicate that it is the ability of rapamycin to inhibit mTOR activation that is responsible for promoting anergy in the presence of costimulation. Therefore, mTOR is required for T cell activation, and TCR engagement in the setting of mTOR inhibition results in anergy.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Rapamycin promotes T cell anergy by inhibiting mTOR. A, Overexpression of RR-mTOR leads to S6K-1 phosphorylation at Thr421/Ser424 even in the presence of rapamycin. Jurkat cells were transfected with empty lenti-vector (EV), lenti-WT-mTOR (WT), or lenti-RR-mTOR (RR), and then treated with rapamycin at 0, 30, and 100 nM for 2 h. Phosphorylation of S6K-1 was analyzed by Western blot. B and C, Overexpression of RR-mTOR prevents rapamycin-induced anergy. The 5C.C7 T cells were infected with WT- or RR-mTOR lentivirus, and stimulated with anti-CD3 plus anti-CD28 (1 + 2), or anti-CD3 plus anti-CD28 plus rapamycin (1 + 2 + R, 30 or 100 nM). After a 5-day rest, the cells were rechallenged with APCs plus PCC peptide. IL-2 production (B) and proliferation (C) upon rechallenge were assessed. Data are representative of three independent experiments.

In vitro, signals 1 and 2 can be precisely controlled. In vivo, however, the induction of T cell activation vs tolerance is most likely determined by the integration of multiple positive and negative accessory signals. To test the hypothesis that mTOR activation plays a critical role in integrating these signals in vivo, we adopted a well-established in vivo peptide-induced anergy model (28). Briefly, CFSE-labeled 5C.C7 T cells (CD4⁺V3⁺) were adoptively transferred into syngeneic B10.A hosts (day 0). The next day, the mice were challenged with soluble PCC peptide (to induce anergy; tolerant) or PCC peptide plus LPS (to promote activation; primed). Also, a cohort of the primed mice received daily rapamycin beginning on day 0 (primed plus R). On day 4, T cells were harvested, and expansion of the transferred clonotypic 5C.C7 cells was analyzed by CFSE dilution (Fig. 6A). First, in the absence of Ag (naive), there was very little proliferation of the clonotypic cell in vivo (R1 gate). In contrast, there was marked proliferation of the cells transferred into the primed mice. Importantly, even though there was less proliferation, essentially all of the clonotypic cells in the tolerant and primed plus R mice proliferated at least once (R2 gate), indicating that essentially 100% of the cells had seen Ag. As seen in Fig. 6B, when equal numbers of clonotypic cells were rechallenged in vitro with APCs plus PCC peptide, the cells from mice treated with peptide alone (tolerant) were hyporesponsive compared with the cells from peptide plus LPS-treated mice (primed). Additionally, as is the case with our in vitro model, the cells that were given full activation in the presence of rapamycin (primed plus R) were rendered anergic. These data support the notion that in vivo TCR engagement in the setting of mTOR inhibition also leads to anergy.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. TCR engagement in the absence of mTOR activity leads to anergy in vivo. A, Adoptively transferred 5C.C7 T cells encountered Ags in vivo. CFSE-labeled 5C.C7 T cells (CD4+V3+) were adoptively transferred into syngeneic B10.A hosts. The mice were then challenged with soluble PCC peptide (Tolerant), PCC peptide plus LPS (Primed), or PCC peptide plus LPS plus rapamycin (Primed + R). Mice that received 5C.C7 cells only (Naive) were used as controls. Division of the transferred 5C.C7 cells was analyzed by CFSE dilution 4 days later. R1 represents clonotypic 5C.C7 cells that did not proliferate in vivo (CFSE high). R2 represents 5C.C7 cells that expanded in response to the Ag challenge in vivo (CFSE low). Percentages of the cells in each gate are listed. B, Rapamycin induces anergy in vivo. CFSE low 5C.C7 T cells from the conditions depicted above were sorted and rechallenged in vitro with APCs plus PCC peptide, and IL-2 production was assessed. C, CD71 surface expression is up-regulated in activated T cell in vivo. The cell surface expression of CD71 on 5C.C7 CFSE low T cells (gate R2) was analyzed by FACS. D, CD71 surface expression distinguishes activated from anergic cells. The 5C.C7 CFSE low T cells (gate R2) from the tolerant and primed mice were sorted based on the surface expression levels of CD71, as indicated in C (labeled as Primed H, Primed L, Tolerant H, and Tolerant L, respectively), and rechallenged in vitro with APCs plus PCC peptide; IL-2 production was analyzed. Data are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The relationship between cell cycle blockade and anergy induction is complex. Several groups have proposed that the cell cycle inhibitor p27^Kip1 plays a role in promoting anergy, whereas in other models, including our own, this does not appear to be the case (9, 10, 12, 14, 29, 30). Using the novel cyclophilin ligand SFA, we showed that G₁ phase arrest does not necessarily lead to anergy (Fig. 1) (13). Furthermore, IL-7-mediated proliferation of anergic cells did not reverse anergy, suggesting that cell cycle progression alone is not sufficient for anergy reversal either. Interestingly, at the biochemical level, it is the activity of mTOR as measured by phosphorylation of S6K-1 at Thr⁴²¹/Ser⁴²⁴ that correlates best with the T cell function in term of activation vs anergy. In this regard, it will be of interest to determine whether mTOR activity is inhibited in models of anergy for which p27^Kip1 plays an important role.

The fact that T cells overexpressing RR-mTOR were resistant to anergy induction provides evidence that the underlying mechanism by which rapamycin promotes anergy is dependent on its ability to inhibit mTOR. This pharmacogenetic approach clearly identifies mTOR activity as playing a role in the decision between full activation vs tolerance. In addition, using a well-established mouse anergy model, we demonstrated that mTOR inhibition by rapamycin also promotes anergy in vivo and that by using cell surface expression of CD71 as a surrogate for mTOR activity we can isolate cells that underwent productive stimulation vs nonproductive stimulation in vivo.

The outcome of Ag recognition is not dictated by the nature of peptide-TCR interaction (signal 1), but rather the context in which this interaction occurs. In the inflammatory milieu, APCs will up-regulate costimulatory molecules, which in turn promote full T cell activation. However, even in this setting, the outcome of TCR engagement will be influenced by cytokine and chemokine stimulation as well as the concomitant engagement of negative receptors such as CTLA-4, PD-1, and the potential presence of regulatory T cells (31). Therefore, just as TOR plays a critical role in sensing signals from growth factors, nutrients, and energy, we propose that mTOR senses the environment for the presence and absence of danger (Fig. 7). In this fashion, mTOR plays a central role in integrating both stimulating and inhibitory signals to execute the appropriate T cell response. We propose that signal 2 represents the net sum of positive and negative signals, which is integrated by mTOR. Based on this model, we predict that signaling pathways that promote mTOR activation (as measured by phosphorylation of S6K-1 at Thr⁴²¹/Ser⁴²⁴) will also promote T cell activation, whereas those that inhibit mTOR will promote anergy.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. A model for the role of mTOR in regulating T cell activation vs tolerance. In mammalian cells, mTOR integrates environmental cues in terms of nutrients, energy, and growth factors, and directs cell growth and proliferation. In the presence of these factors, mTOR is activated and promotes an increase in protein synthesis, cell size, and proliferation (upper left). In contrast, in the absence of these factors, mTOR is inactive, leading to a decrease in protein synthesis, cell size, and proliferation (upper right). By analogy, we propose that in T cells, mTOR integrates environmental cues in terms of the presence or absence of danger. In the setting of danger such as an infection, Ags are presented by activated APCs (lower left). In this context, TCR engagement occurs concomitant with mTOR activation, and hence, a full immune response ensues; signal 1 plus mTOR activation leads to full T cell activation. Alternatively, if Ags (perhaps self peptides) are presented by resting APCs in the absence of danger (lower right), mTOR remains inactive. As a result, the T cells are rendered tolerant; signal 1 in the absence of mTOR activation leads to tolerance.

Finally, our findings have practical implications in terms of developing novel strategies to treat autoimmune disorders and the prevention of graft rejection. Unlike cyclosporin A (CSA) and FK506, rapamycin does not inhibit TCR signaling, and therefore is not a very potent single agent immunosuppressant. However, the ability of rapamycin to promote tolerance makes it a potentially powerful component of an immunosuppressive regimen. Currently, solid organ transplant patients must remain on CSA for life because, although CSA prevents rejection, it also prevents tolerance induction. In experimental models, CSA blocks rapamycin-induced tolerance (35). However, regimens combining noncalcineurin inhibitors (such as costimulatory blockade) with mTOR inhibitors might be able to promote tolerance such that patients are not destined to lifelong immunosuppressive therapy (35, 36). To this end, we have used rapamycin in a mouse model of stem cell transplantation to promote long-term tolerance in the absence of long-term immunosuppression (37).

	Acknowledgments

 
We thank Drs. Xiaobing Yu and Jie Chen for the lentivirus and mTOR constructs; James Flook for high-speed flow sorting; and Dr. Jun O. Liu, Hyam I. Levitsky, Charles G. Drake, Christopher J. Gamper, and Christian F. Meyer for critical review of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants; National Institute of Allergy and Infection Diseases Grant 5K22AI1773 and National Cancer Institute Grant RO1CA98109.

² Address correspondence and reprint requests to Dr. Jonathan D. Powell, Department of Oncology-Immunology/Hematopoiesis, Johns Hopkins School of Medicine, 1650 Orleans Street, CRB 443, Baltimore, MD 21231. E-mail address: Poweljo{at}jhmi.edu

³ Abbreviations used in this paper: mTOR, mammalian target of rapamycin; CSA, cyclosporin A; PCC, pigeon cytochrome c; RR-mTOR, rapamycin-resistant mTOR; S6K-1, S6 kinase-1; SFA, sanglifehrin A; WT-mTOR, wild-type mTOR.

Received for publication May 19, 2006. Accepted for publication November 29, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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