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Article
Nature Immunology - 7, 1166 - 1173 (2006)
Published online: 8 October 2006; Corrected online: 03 November 2006 | doi:10.1038/ni1394

There is an Erratum (December 2006) associated with this Article.

T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-alpha

Yuanyuan Zha1, 4, Reinhard Marks1, 3, 4, Allen W Ho1, 4, Amy C Peterson1, Sujit Janardhan1, Ian Brown1, Kesavannair Praveen1, Stacey Stang2, James C Stone2 & Thomas F Gajewski1

1 Departments of Pathology and Medicine, Section of Hematology and Oncology, University of Chicago, Chicago, Illinois 60637 USA.

2 Department of Biochemistry, The University of Alberta, Edmonton, Alberta T6G2E1, Canada.

3 Present address: Department of Hematology and Oncology, University Hospital Freiberg, 79106 Freiberg, Germany.

4 These authors contributed equally to this work.

Correspondence should be addressed to Thomas F Gajewski tgajewsk@medicine.bsd.uchicago.edu

T cell anergy has been correlated with defective signaling by the GTPase Ras, but causal and mechanistic data linking defective Ras activity with T cell anergy are lacking. Here we used adenoviral transduction to genetically manipulate nonproliferating T cells and show that active Ras restored interleukin 2 production and mitogen-activated protein kinase signaling in T cells that were made anergic in vitro or in vivo. Diacylglycerol kinases (DGKs), which negatively regulate Ras activity, were upregulated in anergic T cells, and a DGK inhibitor restored interleukin 2 production in anergic T cells. Both anergy and DGK-alpha overexpression were associated with defective translocation of the Ras guanine nucleotide–exchange factor RasGRP1 to the plasma membrane. Our data support a causal function for excess DGK activity and defective Ras signaling in T cell anergy.
Engagement of the T cell receptor (TCR) in the absence of CD28 costimulation can result in a long-term hyporesponsive state called 'clonal anergy'1. Anergic T cells have defective production of interleukin 2 (IL-2) and reduced proliferation after restimulation via the TCR and CD28. Anergy induction depends on calcium, is mimicked by iomomycin and is prevented by cyclosporin A, indicating that anergy is induced in conditions in which signaling by calcium and the transcription factor NFAT are disproportionately increased over other signals. Anergy may represent one mechanism of peripheral tolerance2 and has been reported to occur in the setting of nonproductive antitumor immunity in vivo3. Thus, understanding the regulation of induced T cell hyporesponsiveness may enable the manipulation of immune responses to favor tolerance over activation and may have broad potential applications in the therapy of disease states associated with immune dysregulation.

Molecular alterations that correlate with defective IL-2 production in response to TCR and CD28 engagement have been evaluated in some detail4, mainly in T helper type 1 (TH1) clones. Anergic T cells have defective activation of the GTPase Ras, which is associated with diminished activation of the mitogen-activated protein (MAP) kinases Erk and Jnk5, 6 as well as blunted transactivation of the AP-1 transcription factor7. The ability of dominant negative Ras to inhibit IL-2 promoter activity in T cell tumor lines8, coupled with the block in thymic development seen in mice transgenic for dominant negative Ras9, support the idea of an important function for Ras signaling in TCR-mediated T cell activation. Nevertheless, whether impaired Ras activation is sufficient to explain the complete phenotype of anergic T cells has not been determined.

In part, the lack of mechanistic data regarding Ras and anergy is due to the technical limitations of strategies for in vitro genetic manipulation of T cells. In particular, a key experiment would require the introduction of constitutively active Ras into T cells already made anergic and determination of whether MAP kinase activation and IL-2 production could be restored. Retroviral transduction requires cell proliferation, which is not possible with growth-arrested anergic T cells.

Here we used T cells from mice transgenic for the coxsackie and adenovirus receptor (CAR), which in a quiescent state can be transduced with adenovirus vectors10. We found that constitutively active Ras restored cytokine production in anergic T cells. Those data demonstrated a causal link between defective Ras signaling and T cell anergy. We assessed candidate mechanisms to explain defective Ras activation in anergic T cells and used gene array analysis to identify diacylglycerol kinases (DGKs) as anergy-associated molecules that act 'upstream' of and negatively regulate Ras in anergic T cells.

Results
Active Ras reverses the anergic TH1 cell phenotype
The well defined biochemical alterations occurring during T cell anergy have been described mainly with TH1 cells as a model system. Anergic TH1 cells have blunted activation of Ras and of the 'distal' MAP kinases Erk and Jnk. To determine whether defective Ras signaling contributes to TH1 cell anergy, we introduced constitutively active Ras into anergic TH1 cells and assessed whether IL-2 production and MAP kinase activation were restored. To circumvent technologic limitations that have precluded genetic manipulation of nonproliferating T cell populations, we used T cells from CAR-transgenic mice ('CAR mice'), which in a quiescent state can be transduced with high efficiency with adenovirus vectors10. In experiments to design viral vectors suitable for expression in primary T cells, a series of promoters was screened, and the human ubiquitin C promoter11 was found to induce expression 1–2 logs greater in quiescent T cells than did other common promoters12. Ovalbumin-specific TH1 clones were generated from CAR mice10. Here, we transduced control and anergic CAR TH1 cells in a similar way with an adenovirus vector encoding green fluorescent protein (GFP) driven by the ubiquitin C promoter (Fig. 1a). An empty control adenovirus did not 'transfer' green fluorescence (data not shown). Transduction with recombinant adenovirus encoding the constitutively active Ras mutant Ras61L under control of this promoter resulted in substantial expression of the H-R as isoform in CAR TH1 cells (Fig. 1b).

Figure 1. Constitutively active Ras restores IL-2 production in CAR TH1 cells made anergic in vitro.
Figure 1 thumbnail

(a) Flow cytometry of control and anergic CAR TH1 cells transduced with adenovirus vector encoding GFP. Similar results were obtained in at least two experiments. (b) Immunoblot analysis of H-Ras in CAR TH1 cells transduced with empty vector or with adenovirus vector encoding Ras61L. Similar results were obtained in five experiments. (c) ELISA of IL-2 in supernatants of anergic CAR TH1 cells left untransduced or transduced with empty vector or with a vector encoding Ras61L, then stimulated with 'empty' beads (Beads) or with beads coated with anti-CD3 and anti-CD28 (CD3 + CD28) or with PMA plus ionomycin (P + I) and analyzed at 18 h. Control, nonanergic cells. Results are representative of at least five experiments.



Full FigureFull Figure and legend (15K)
Anergy has been induced in TH1 cells with a variety of stimuli that engage the TCR complex in the absence of CD28 costimulation1. As a first approach to induce and assess anergy, we stimulated CAR TH1 cells with plate-bound antibody to CD3 (anti-CD3), allowed the TH1 cells to 'rest' and restimulated them with anti-CD3 plus anti-CD28 or with phorbol 12-myristate 13-acetate (PMA) plus ionomycin. TH1 cells treated in that way produced less IL-2 after restimulation with anti-CD3 plus anti-CD28 but produced normal quantities of IL-2 after restimulation with PMA and ionomycin (Fig. 1c). Biochemical analysis showed less phosphorylation of Erk and Jnk after restimulation with anti-CD3 and anti-CD28 (Fig. 2a). These results were consistent with published observations of anergic TH1 cells4.

Figure 2. Constitutively active Ras restores MAP kinase signaling and IL-2 promoter activity in TH1 cells made anergic in vitro.
Figure 2 thumbnail

(a) Immunoblot analysis of phosphorylated Erk (p-Erk) and total Erk (left) or phosphorylated Jnk (p-Jnk) and total Jnk (right) in CAR TH1 cells left untransduced (None) or transduced with empty vector or with a vector encoding Ras61L, then left unstimulated (–) or stimulated (+) for 20 min with beads coated with anti-CD3 and anti-CD28 (below lanes). Control, nonanergic CAR TH1 cells. (b) Luciferase activity of cells transduced as described in a and then stimulated 20 h with 'empty' beads or with beads coated with anti-CD3 and anti-CD28. Similar results were obtained in two (a) or three (b) experiments (error bars (b), s.d.).



Full FigureFull Figure and legend (23K)
To determine whether active Ras would restore anergic TH1 cell activity, we transduced anergic CAR TH1 cells with empty adenovirus or with adenovirus encoding Ras61L. The introduction of Ras61L but not the empty adenovirus resulted in substantial production of IL-2 and phosphorylation of Erk and Jnk in anergic TH1 cells restimulated with anti-CD3 and anti-CD28 (Figs. 1c and 2a). That cytokine production was not constitutive but required TCR engagement, consistent with data indicating that Ras is not sufficient to induce all signals leading to cytokine gene expression. That result was not due to massive overexpression of active Ras, as IL-2 production was restored with as little virus as a multiplicity of infection of one, which resulted in Ras expression that barely exceeded total endogenous Ras expression (Supplementary Fig. 1 online). To determine whether the restoration of IL-2 production by Ras61L occurred at the level of Il2 transcription, we generated TH1 clones from CAR mice that we crossed with mice expressing a transgene containing a luciferase construct driven by the Il2 promoter ('IL-2–Luc' mice). We rendered the TH1 clones from the resultant 'CAR IL-2–Luc' progeny anergic and transduced them with empty or Ras61L adenovirus. Anergic CAR IL-2–Luc TH1 cells had less Il2 promoter activity, which was reversed after the introduction of Ras61L (Fig. 2b). These results demonstrated that active Ras can restore transcription of Il2 and phosphorylation of Erk and Jnk in TH1 cells already made anergic.

To determine whether active Ras could restore the function of T cells made anergic in vivo and to assess whether this manipulation was also effective with CD8+ T cells, we crossed CAR mice with recombination-activating gene 2–deficient (Rag2-/-) mice expressing the major histocompatibility class I–restricted 2C TCR transgene ('2C Rag2-/-' mice). T cells from progeny of that cross ('CAR 2C Rag2-/-' mice) had high expression of CAR and were transduced with an efficiency of 80–90% with adenovirus vector encoding GFP (data not shown). Repeated administration of the H2-Kb-binding 2C TCR cognate antigenic peptide SIYRYYGL to 2C TCR–transgenic mice results in T cell hyporesponsiveness, consistent with anergy induction13. We confirmed that CD8+ T cells from CAR 2C Rag2-/- mice treated with soluble SIYRYYGL peptide had defective IL-2 production and reduced Erk phosphorylation after in vitro restimulation with P815 tumor cells expressing the specific alloantigen Ld and transfected to express B7-1 ('P815.B71' cells) or after in vitro restimulation with anti-CD3 plus anti-CD28 (Fig. 3a). However, transduction with adenovirus encoding Ras61L before in vitro restimulation restored IL-2 production and Erk phosphorylation to CAR 2C Rag2-/- CD8+ T cells made anergic in vivo (Fig. 3). These data indicated that defective Ras signaling is causally linked to the anergic state of CD8+ T cells rendered hyporesponsive in vivo.

Figure 3. Constitutively active Ras restores IL-2 production and MAP kinase activation in CAR CD8+ 2C T cells made anergic in vivo.
Figure 3 thumbnail

CAR 2C Rag2-/- mice were treated with SIYRYYGL peptide to induce anergy, then CD8+ T cells were collected and were left untransduced or were transduced with empty vector or with a vector encoding Ras61L. (a) ELISA of IL-2 in supernatants of cells stimulated for 18 h with 'empty' beads, with P815.B71 cells, or with beads coated with anti-CD3 and anti-CD28 or with PMA plus ionomycin. Control, CD8+ cells from PBS-treated CAR 2C Rag2-/- mice. (b) Immunoblot analysis of phosphorylated Erk and total Erk in lysates of cells left unstimulated (-) or stimulated (+) for 20 min with beads coated with anti-CD3 and anti-CD28 (below lanes). Similar results were obtained in two experiments.



Full FigureFull Figure and legend (17K)
Anergy independent of the adaptor CrkL and ligase Cbl
Having confirmed the importance of defective Ras signaling in T cell anergy, we sought to identify the mechanism by which Ras activation is blunted during anergy. The anergic state is thought to require the production of a new protein, as it is prevented by the presence of cycloheximide14, and heterokaryon fusion experiments have shown that anergy is mediated in part by a dominant suppressive factor15. One hypothesis proposes that anergy is mediated by increased recruitment of complexes containing the CrkL adaptor protein and the C3G guanine nucleotide–releasing factor (which promotes guanosine triphosphate exchange of the alternative Ras family GTPase Rap1) to the plasma membrane via the Cbl E3 ubiquitin ligase16. Rap1 activation antagonizes Ras signaling17 and mimics an anergic phenotype in Jurkat T cells. However, Rap1 also has other effects, including the stimulation of cell adhesion18. We confirmed that TH1 cells made anergic with plate-bound anti-CD3 contained substantial C3G as well as C3G-CrkL complexes (Supplementary Fig. 2 online).

To assess whether that increase in CrkL-C3G complexes was required for TH1 cell anergy, we analyzed TH1 clones from CrkL-deficient mice19. We confirmed the absence of CrkL by immunoblot analysis (Supplementary Fig. 2), and found that CrkL-deficient TH1 cells produced IL-2 (Fig. 4a) and interferon-gamma (data not shown) after stimulation with anti-CD3 and anti-CD28. However, like wild-type TH1 cells, CrkL-deficient TH1 cells were rendered anergic after stimulation with immobilized anti-CD3, as indicated by reduced IL-2 production (Fig. 4a) and blunted Erk phosphorylation (Supplementary Fig. 2). These results demonstrated that CrkL is not absolutely required for the induction or maintenance of T cell anergy.

Figure 4. CrkL and Cbl function are not required for T cell anergy.
Figure 4 thumbnail

(a) ELISA of IL-2 production by wild-type (+/+) TH1 clones (CWG11 and PGL10) and CrkL-deficient (-/-) TH1 clones (CKG5 and CKG3) left unmanipulated (Control) or made anergic with anti-CD3, then stimulated with beads coated with anti-CD3 and anti-CD28 and analyzed after 18 h. (b) ELISA of IL-2 production by CAR TH1 cells left unmanipulated or made anergic with anti-CD3, then transduced with empty vector or with adenovirus encoding dominant negative Cbl (DN Cbl) and stimulated with 'empty' beads or beads coated with anti-CD3 and anti-CD28. Similar results were obtained in two (a) or three (b) experiments (error bars (b), s.d.).



Full FigureFull Figure and legend (17K)
T cells from Cbl-b-deficient mice can be activated independently of CD28 stimulation and are relatively resistant to anergy20. To determine whether inhibition of Cbl-b function could reverse established anergy, we used the viral Cbl structure to design an adenovirus vector containing a dominant negative Cbl construct21. The truncated Cbl protein encoded by this construct is expected to bind to 'upstream' signaling proteins but lacks the RING finger domain responsible for E3 ubiquitin ligase activity. Nonanergic CAR TH1 cells transduced with that dominant negative Cbl adenovirus expressed Cbl in excess of endogenous Cbl, as detected by immunoblot, and produced more IL-2 (Supplementary Fig. 3 online). However, expression of dominant negative Cbl did not restore IL-2 production in anergic CAR TH1 cells (Fig. 4b). These results indicated that Cbl-independent mechanisms of blunting Ras activation exist in anergic TH1 cells.

Gene expression in anergic TH1 cells
To identify proteins that might negatively regulate Ras activation in anergic TH1 cells, we used gene chips containing sequences from 11,000 mouse genes to compare the gene expression profiles of resting control and anergic TH1 cells. Anergic TH1 cells had 135 genes with expression twofold or higher than that in resting TH1 cells (Supplementary Note online). Among those were two genes encoding molecules that could theoretically negatively regulate Ras activity: a Ras-GAP–like molecule and DGK-alpha. Confirmatory analysis by semiquantitative RT-PCR showed that DGK-alpha mRNA but not the Ras-GAP–like transcript was upregulated in TH1 cells rendered anergic by plate-bound anti-CD3 (Fig. 5a and data not shown).

Figure 5. Expression of DGK-alpha is upregulated in anergic TH1 cells.
Figure 5 thumbnail

(a) Semiquantitative RT-PCR of transcripts encoding DGK-alpha (Dgka) or beta-actin (Actb) in pGL10 cells (normal mouse TH1 clone specific for ovalbumin B) left unmanipulated (C) or made anergic with anti-CD3 (A) or ionomycin (I) or treated with ionomycin and cyclosporin A (I + Cy). Wedges indicate serial threefold dilutions of cDNA. Similar results were obtained in two experiments. (b) Immunoblot analysis of DGK-alpha and RasGRP1 in control (nonanergic) and anergic pGL10 cells. (c) Densitometric quantification of DGK-alpha immunoblot data in b, obtained in three experiments (error bars, s.d.).



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DGK molecules phosphorylate diacylglycerol to generate phosphatidic acid, thus reducing the availability of diacylglycerol. Diacylglycerol activates 'downstream' effector proteins such as the alternative Ras guanine nucleotide–exchange factor RasGRP1, which is activated after TCR ligation22, 23, 24 and has been associated with regulation by DGKs25. Thus, increased expression of DGK molecules might be expected to suppress RasGRP1-mediated Ras activation26.

To 'solidify' the correlation between increased DGK expression and anergy, we assessed additional cellular parameters. Anergy is induced by an increase in intracellular calcium alone and is prevented by treatment with cyclosporin A14. Expression of DGK-alpha mRNA was upregulated by treatment with ionomycin, and this upregulation was prevented in the presence of cyclosporin A (Fig. 5a). Expression of the 'housekeeping' gene encoding beta-actin remained relatively constant in those treatment conditions. This secondary screen thus indicated that the key signaling conditions favoring anergy induction also favor upregulation of DGK mRNA.

At least two DGK isoforms, DGK-zeta and DGK-alpha, are expressed in T cells27, 28. Immunoblot with isoform-specific antibodies showed that DGK-alpha expression was approximately five- to tenfold higher in anergic than in control TH1 cells (Fig. 5b,c), whereas DGK-zeta protein expression was approximately twofold higher (data not shown). Thus, expression of DGK-alpha and DGK-zeta correlates with the anergic state of TH1 cells. Immunoblot analysis confirmed that RasGRP1 expression was not lower in anergic TH1 cells (Fig. 5b).

Defective membrane recruitment of RasGRP1
If TH1 cell anergy were caused by increased DGK expression and diminished diacylglycerol-dependent activation of Ras, the anergic state would be expected to be associated with defective translocation of RasGRP1 to the plasma membrane. P815.B71 cells, which express the alloantigen Ld and the CD28 ligand B7-1 (ref. 29), stimulate 2C T cells. Signaling events occurring in such stimulated 2C T cells can be visualized with immunofluorescence techniques. Therefore, we used that system to assess RasGRP1 localization. We stained 2C T cell–P815.B71 conjugates with anti-RasGRP1 and anti-talin (control) as well as appropriate fluorochrome-coupled secondary antibodies. We also prelabeled the P815.B71 cells with the cell tracker CMAC to distinguish them from the T cells. RasGRP1 was present mainly in the cytosol of unconjugated unstimulated 2C T cells (data not shown). In 2C T cells conjugated with P815.B71 cells, most RasGRP1 was detected at the T cell–antigen-presenting cell (APC) interface (Fig. 6a). In contrast, 2C T cells rendered anergic by the in vivo administration of soluble peptide antigen had defective recruitment of RasGRP1 to the T cell–APC interface. We quantified those data in terms of the fraction of conjugates with that pattern as well as by the mean pixel intensity of RasGRP1 staining at the T cell–APC interface relative to that of the T cell cytosol (Fig. 6b,c). Thus, T cell anergy is associated with defective RasGRP1 recruitment to the T cell–APC contact site.

Figure 6. Defective localization of RasGRP1 to the plasma membrane in anergic 2C T cells.
Figure 6 thumbnail

In vivo administration of soluble peptide antigen was used to render 2C Rag2-/- cells anergic; in vitro–primed 2C Rag2-/- T cells serve as a control. Conjugates were established with CMAC-labeled P815.B71 cells, followed by staining with anti-RasGRP1 (green) and anti-talin (red). (a) Representative images of conjugates containing control (top) and anergic (bottom) 2C Rag2-/- T cells. Far left, differential interference contrast images of the cells at right. Original magnification, times 630. (b) Proportion of conjugates with membrane recruitment of RasGRP1. (c) Pixel intensity of RasGRP1 at the plasma membrane of the T cell–APC interface relative to that in the cytosol, for 15 conjugate images (error bars, s.d.). Similar data were obtained in two independent experiments.



Full FigureFull Figure and legend (30K)
Causative function of DGK in T cell anergy
Next we sought to determine whether overexpression of DGK molecules was sufficient to induce an anergic phenotype in resting TH1 cells. For this, we constructed adenovirus vectors encoding DGK-alpha and DGK-zeta. CAR TH1 cells transduced with the vector encoding DGK-alpha contained DGK-alpha protein (Fig. 7a) and produced less IL-2 in response to stimulation with anti-CD3 and anti-CD28 (Fig. 7b). In addition, overexpression of DGK-alpha suppressed phosphorylation of Erk and Jnk induced by anti-CD3 and anti-CD28 (Fig. 7c). Transduction with DGK-zeta resulted in minimal effects on the production of IL-2 and phosphorylation of Erk and Jnk (data not shown). A lower multiplicity of infection of adenovirus, which resulted in expression of DGK-alpha only twofold higher than endogenous expression (in the same range of the upregulated DGK-alpha expression in anergic TH1 cells), was sufficient to inhibit IL-2 production (Supplementary Fig. 4 online). To confirm that the inhibition of IL-2 production was regulated at the level of the Il2 promoter, we transduced and stimulated CAR IL-2–Luc TH1 cells in a similar way. Overexpression of DGK-alpha inhibited Il2 promoter activity (Fig. 7d). In addition to its effects on Il2 transcription, DGK-alpha overexpression resulted in impaired RasGRP1 plasma membrane localization in 2C cells conjugated with P815.B71 cells (Fig. 8). These results collectively suggested that overexpression of DGK-alpha can mimic the functional and biochemical characteristics of the anergic state.

Figure 7. Overexpression of DGK-alpha results in a state resembling anergy.
Figure 7 thumbnail

(a) Immunoblot analysis with anti-Myc (top) and anti-Erk (bottom) of CAR TH1 cells transduced with empty vector or with vector encoding Myc-tagged DGK-alpha. (b) ELISA of IL-2 in supernatants of transduced CAR TH1 cells stimulated for 18 h with empty beads (Control) or with beads coated with anti-CD3 and anti-CD28 or with PMA plus ionomycin. (c) Immunoblot analysis of phosphorylated Erk and Jnk and of DGK-alpha (assessed with anti-Myc) in transduced CAR TH1 cells stimulated for 20 min (above lanes); blots were stripped and reprobed to measure total Erk and total Jnk. (d) Luciferase activity of CAR IL-2–Luc TH1 cells transduced and stimulated as described in b. Control, cells stimulated with empty beads. Similar results were obtained in three experiments (error bars (b,d), s.d.).



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Figure 8. Overexpression of DGK-alpha results in diminished RasGRP1 plasma membrane translocation.
Figure 8 thumbnail

CAR 2C Rag2-/- T cells were primed in vitro and were transduced with empty vector or with vector encoding DGK-alpha. Conjugates were established with CMAC-labeled P815.B71 cells, followed by staining with anti-RasGRP1 (green) and anti-talin (red). (a) Representative images of conjugates containing 2C Rag2-/- T cells transduced with empty vector (top) or vector encoding DGK-alpha (bottom). Original magnification, times 00. (b) Immunoblot analysis of Myc-tagged DGK-alpha (Myc) and Erk in transduced cells. MOI, multiplicity of infection. (c) Pixel intensity of RasGRP1 at the plasma membrane of the T cell–APC interface relative to that in the cytosol, for 15 conjugate images (error bars, s.d.). Similar data were obtained in two independent experiments.



Full FigureFull Figure and legend (31K)
If increased DGK activity promotes diminished Ras activation and IL-2 production in anergic TH1 cells, DGK-alpha inhibition should result in augmented IL-2 production by anergic TH1 cells. To explore that possibility, we used TH1 cells rendered anergic by stimulation with immobilized anti-CD3 and treated them with a pharmacologic inhibitor of DGK activity (DGK I)30. Treatment with DGK I resulted in substantial dose-dependent recovery of IL-2 production by anergic TH1 cells stimulated with anti-CD3 and anti-CD28 (Fig. 9a). The recovery of IL-2 production in various experiments ranged from 21% to 108%, corresponding to a 2.4-fold to a 4.8-fold increase in IL-2 production by anergic TH1 cells. The inhibitor alone did not induce detectable cytokine release (data not shown). To determine whether the same effect would be obtained with T cells made anergic in vivo, we rendered 2C T cells anergic with soluble peptide antigen and treated them with DGK I. The 2C T cells made anergic in vivo also demonstrated substantial recovery of IL-2 production in the presence of DGK I (Fig. 9b). These results suggested a causal function for increased DGK function in mediating T cell anergy.

Figure 9. A DGK inhibitor partially restores IL-2 production by anergic TH1 cells.
Figure 9 thumbnail

(a) ELISA of IL-2 production by CAR TH1 cells left unmanipulated (Control) or made anergic with anti-CD3 (Anergic), then incubated for 25 min in the presence of increasing concentrations of the DGK inhibitor DGK I (below bars) before stimulation overnight with empty beads (–) or with beads coated with anti-CD3 and anti-CD28 (+). (b) ELISA of IL-2 production by 2C Rag2-/- T cells made anergic by in vivo injection of soluble antigenic peptide, then stimulated overnight in vitro in the absence or presence of increasing concentrations of DGK I (below bars). Similar results were obtained in three experiments (error bars, s.d.).



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 Top
Discussion
T cell anergy has been correlated with defective activation of Ras, Erk, Jnk and AP-1. Here we have demonstrated that the introduction of constitutively active Ras restored IL-2 production and MAP kinase activation in T cells made anergic in vitro or in vivo. Those results indicated a causal function for blocked Ras in inducing and/or maintaining a state of anergy in T cells. However, although we assessed several features of anergy with both in vitro and in vivo models of T cell anergy, it is conceivable that there may be subtle differences in the mechanisms by which T cells are rendered hyporesponsive in those two systems. In both models, 'CAR technology' made it possible to transduce nonproliferating anergic T cells and to analyze effects of genetic manipulation on endogenous cytokine production and Il2 promoter activity rather than on reporter constructs introduced by cotransfection.

Reports have suggested that increased recruitment of CrkL-C3G complexes and concomitant Rap1 activation might be responsible for the decreased TCR-induced activation of Ras and MAP kinases and for the reduced production of IL-2 in anergic T cells16. However, CrkL-deficient mice do not demonstrate T cell hyper-responsiveness31, and we have shown here that T cells derived from CrkL-deficient mice were susceptible to anergy induction. In addition, it has been shown that transgenic expression of Rap1 in T cells promotes increased T cell activation through augmented adhesion18. Those results collectively suggest that CrkL-C3G and Rap1 are probably not essential for the induction of T cell anergy, although those molecules could conceivably dampen TCR signaling in other circumstances.

Here we have identified DGK-alpha as an anergy-associated factor capable of negatively regulating TCR-induced Ras activation. The identification of RasGRP1 as a key guanine nucleotide–exchange factor for Ras in T cells revealed a mechanism by which PMA and/or diacylglycerol can activate Ras23, 24. By depleting available diacylglycerol, DGK blocks that route of Ras activation. At least nine isoforms of DGK have been identified, and both DGKalpha and DGKzeta are reported to be expressed in T cells27, 28, 32. Forced overexpression of DGKzeta has been shown to inhibit TCR-induced activation of Ras, Erk and AP-1 in Jurkat cells27, indicating that large amounts of DGKzeta can be sufficient to inhibit T cell activation in some systems. Here we have shown that upregulated expression of DGKalpha and DGKzeta correlated with the anergic state and that a pharmacologic inhibitor of DGK partially derepressed IL-2 production by anergic T cells. The incomplete reversal of anergy noted in some experiments with the DGK inhibitor suggested either that this compound does not completely inhibit all DGK isoforms or that additional molecular mechanisms function in maintaining the anergic state.

The recovery of IL-2 production by anergic T cells with the DGK inhibitor in our study is subject to the general caveats of using pharmacologic inhibitors for mechanistic experiments. However, consistent with our results, T cells derived from DGK-alpha-deficient mice are also relatively resistant to anergy induction (G. Koretzky, personal communication). In addition, T cells from DGKzeta-deficient mice have heightened sensitivity to TCR stimulation33. Those observations support the idea that DGKs have an important negative regulatory function in T cell activation. Further delineation of the inhibitory effect of the various DGK isoforms in TCR signaling may require conditional deletion of the isoforms, alone and in combination, in post-thymic T cell subsets.

Although DGKs in anergic T cells probably act by converting diacylglycerol to phosphatidic acid, we were unable to reproducibly quantify those lipids directly. However, our data showing defective translocation of RasGRP1 to the T cell–APC interface, which is dependent on diacylglycerol, support the idea of that mechanism of action. Still, the possibility of alternative mechanisms of DGK action cannot be excluded. In addition, it is conceivable that DGKs might also be regulated at the level of functional activity in addition to expression, and depletion of diacylglycerol by DGKs could also affect other signaling pathways, such as those driven by protein kinase C isoforms.

Mice deficient in RasGRP1 have been generated by gene targeting24 and have also been characterized as spontaneous mutants34. Although those mice have defects in thymic development, they eventually, paradoxically, develop spontaneous T cell activation and autoimmunity. It is conceivable that the T cells that successfully develop in the absence of RasGRP1 use compensatory TCR signaling pathways that are less influenced by negative regulation than is RasGRP1 by DGKs.

Other gene products have been reported as candidates for contributing to T cell hyporesponsiveness in the anergic state. The transcriptional regulator Tob is upregulated in anergic T cells, and enforced expression of Tob results in diminished IL-2 production35. However, naive T cells also have high expression of Tob and show robust IL-2 production in response to TCR and CD28 ligation. Thus, although signals involving Tob may be important for the negative regulation of T cell activation, Tob expression does not always seem to correlate with an anergic phenotype. GRAIL, an E3 ubiquitin ligase, has been identified as being upregulated in anergic T cells and negatively regulating TCR-triggered cytokine gene expression36. It is conceivable that GRAIL could also antagonize Ras signaling and/or that diminished Ras signaling could promote GRAIL upregulation.

Our results are apparently in contrast to a report showing T cells transduced with a retrovirus encoding active Ras are still rendered hyporesponsive37. However, retroviral transduction requires that the cells be transduced before being made anergic, and it is conceivable that early and constitutive Ras activation might itself contribute to T cell dysregulation. Our experimental approach allowed the introduction of active Ras after anergy was induced and in model systems in which blockade of Ras signaling is known to occur.

In summary, our results have suggested that the blockade in Ras signaling in T cell anergy causally contributes to the hyporesponsive state. We have also identified increased expression of DGK isoforms as contributing to the hyporesponsive status of anergic T cells. The development of selective pharmacological agents that inhibit the activity of individual DGK isoforms could lead to new ways to augment T cell responses in vivo.

 Top
Methods
Mice and cell lines.
Mice expressing the extracellular domain of CAR10, 2C TCR–transgenic Rag2-/- mice38 and IL-2–Luc mice39 have been described. Mice were maintained in specific pathogen–free conditions in a barrier facility at the University of Chicago (Chicago, Illinois) according to institutional guidelines. TH1 clones were generated in the Gajewski laboratory by immunization with ovalbumin as described10 and were maintained by weekly passage as reported40. P815.B71 cells were maintained as described41.

Induction of T cell anergy.
TH1 cells were made anergic by stimulation for 24–48 h with plate-bound anti-CD3, were collected and were allowed to 'rest' for 24–72 h in culture medium alone as described14. Alternatively, anergy was induced by exposure to ionomycin (0.5 muM) or the presence of cyclosporin A (50 ng/ml)14. In vivo anergy induction of 2C T cells by the administration of SIYRYYGL peptide was done as described13.

Adenovirus vectors.
The cDNA encoding Ras61L was provided by F. Fitch (University of Chicago, Chicago, Illinois). The dominant negative Cbl construct was generated by RT-PCR with cDNA from TH1 clones as a template and the following primers (upper case, restriction enzyme sequences; underlining, Myc tag sequence): 5'-GGGGTACCatggagcagaaactcatctctgaagaggatctggccggcaacgtgaagaaga-3' (forward) and 5'-ATAGTTTAGCGGCCGCtcaatcttgaggagttggtt cacataa-3' (reverse). The cDNA encoding DGK-alpha was a gift from M. Topham (University of Utah, Salt Lake City, Utah) and was used as a template to introduce an N-terminal Myc epitope tag by PCR. The sequences of all PCR products were confirmed before subcloning. Construction of recombinant adenovirus vectors was done with a two-cosmid system that has been described42.

Adenoviral transduction of CAR T cells.
TH1 clones were purified from passage cultures by Ficoll-Hypaque centrifugation. Primary CAR 2C Rag2-/- CD8+ T cells were isolated from splenocytes by negative selection with magnetic beads and antibody 'cocktails' (Stem Cell Technologies). CAR TH1 cells were transduced with adenovirus vectors at high cell density (1 times 107 cells/ml) in DMEM containing 2% (volume/volume) FCS and were incubated for 1 h at 37 °C, followed by an overnight 'rest' at 37 °C in DMEM containing 5% (volume/volume) FCS at low cell density (4 times 105 cells/ml).

Flow cytometry.
TH1 cells transduced with adenovirus vector encoding GFP were analyzed with a FACScan (BD Biosciences). A total of 1 times 104 events were acquired, and data were analyzed with CellQuest software (BD Biosciences).

T cell activation.
T cells were activated with beads (Dynal) coated with anti-CD3 (145-2C11) and anti-CD28 (PV1) as described43. Cytokines in the supernatants of 1 times 105 TH1 cells stimulated for 18 h at 37 °C in microtiter plates were measured by enzyme-linked immunosorbent assay (ELISA) with antibody pairs obtained from BD Pharmingen. As a control, TH1 cells were stimulated with PMA (50 ng/ml) and ionomycin (0.5 muM). In some experiments, TH1 cells were incubated in the presence of DGK I (Sigma).

Luciferase assays.
CAR IL-2–Luc TH1 clones were transduced with vectors, were stimulated for 20 h and were resuspended in serum-free DMEM in luminometer cuvettes (BD Biosciences). An equal volume of Bright-Glo luciferase assay reagent (Promega) was added to each sample, followed by thorough mixing. After 2 min, samples were analyzed with a monolight 2010 Luminometer (BD Biosciences).

Immunoblot.
Cells were lysed in 0.5% (volume/volume) Triton X-100 lysis buffer and immunoblot analysis was done as described43. Immunoprecipitation with anti-CrkL or control rabbit antiserum was done as described44. Antibodies to the following were used: phosphorylated Erk (910L; Cell Signaling); phosphorylated Jnk (V7932; Promega); Erk (13-6200; Zymed); Jnk1 (sc-474), H-Ras (sc-35), C3G (sc-869), CrkL (sc-319), RasGRP1 (sc-8430) and DGK-zeta (sc-8722; all from Santa Cruz Biotechnologies); and DGK-alpha (a gift from H. Kanoh, Sapporo Medical University, Sapporo, Japan). Images were scanned, followed by densitometry analysis with UN-SCAN-IT software (Silk Scientific).

Gene expression profiling and RT-PCR.
These procedures are described in the Supplementary Methods online.

Immunofluorescence microscopy.
Analysis of protein localization in 2C T cell–P815.B71 cell conjugates was done as described29. P815.B71 cells were labeled with CMAC (7-amino-4-chloromethylcoumarin) Cell-Tracker Blue (Molecular Probes) and were mixed with equal numbers of anergic or in vitro–primed 2C Rag2-/- T cells. After approximately 8 min, cells were fixed, were made permeable and were stained with anti-GRP1 and anti-talin (Santa Cruz Biotechnologies) and with species-specific secondary antibodies conjugated to fluorescein isothiocyanate or phycoerythrin, respectively. Samples were analyzed with a Zeiss Axiovert 100 microscope, and 15 conjugates were typically assigned scores. Slidebook software (Intelligent Imaging Innovations) was used for image capture and deconvolution analysis. ImageJ 1.36b software (US National Institutes of Health) was used for quantification of pixel intensity.

Statistical methods.
Differences between data sets were analyzed with the two-sided Student's t-test and Excel software.

Note: Supplementary information is available on the Nature Immunology website.

Author contributions
Y.Z. did experiments involving the cloning, transduction and expression of DGK-alpha and DGK-zeta, the effects of the DGK inhibitor, the generation of the dominant negative Cbl adenovirus and experiments associated with it, and participated in writing the manuscript; R.M. developed adenoviruses encoding active Ras and GFP and adenoviral transduction protocols, immunized mice and derived CAR transgenic TH1 clones, and showed that active Ras could restore IL-2 production by anergic TH1 cells; A.W.H. did gene expression profiling of anergic TH1 cells and confirmed the effects of active Ras on anergic T cells and effects of the DGK inhibitor; A.C.P. did all experiments with CrkL-deficient T cells; S.J. and I.B. worked together to accomplish the in vivo anergy experiments with 2C Rag2-/- T cells; K.P. did confocal microscopy of 2C cells conjugated with P815.B71 cells; S.S. and J.C.S. did immunoblot analysis of DGK-alpha and provided experimental guidance; T.F.G. conceived of and directed the project, oversaw all experiments, secured funding and actively participated in manuscript writing.

 Top
Received 16 June 2006; Accepted 22 August 2006; Published online: 8 October 2006.

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Acknowledgments
We thank J. DeGregori for the human ubiquitin C promoter; J. Washington for assistance with mouse breeding; and C. Kao, F. Rivas, R. Shah and S. Bond for experimental assistance. Supported the National Institutes of Health (R01AI7919, P01CA97296 and R21AI59818), the Arthritis Foundation (T.F.G.) and Deutsche Forschungsgemeinschaft (R.M.).

Competing interests statement:  The authors declare that they have no competing financial interests.

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