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Departments of Medicine and Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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B were monitored after cross-linking of the receptor on naive and activated T cells. GITR cross-linking provided costimulation of naive and activated T cells and resulted in activation of MAPKs and NF-B. Although GITR-induced signaling pathways augmented the survival of naive T cells, they were not sufficient to inhibit activation-induced cell death triggered by CD3 cross-linking of activated T cells. Differences in the contributions of GITR to cell survival between naive and activated T cells suggest that the receptor triggers specific pathways depending on the activation state of the T cell. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The development of lymphocytes, including T cells, is grossly normal in GITR-deficient mice (3). However, T cells from GITR-deficient mice display an exaggerated activation phenotype when stimulated with CD3-specific Ab. Compared with GITR-sufficient controls, GITR–/– T cells express higher levels of IL-2, the high affinity IL-2R (CD25), and Fas (CD95), which results in increased proliferative responses and increased susceptibility to activation-induced cell death (AICD). Consistently, lowering GITR expression by antisense mRNA sensitizes cells to cell death, whereas overexpression of GITR increases the resistance of T cell clones to apoptosis induced by anti-CD3 Ab (2). However, recent reports suggest that GITR can increase AICD and potentially trigger cell death through its interaction with the proapoptotic protein Siva (8, 9). These studies point to GITR as a receptor that regulates T cell survival, but the molecular mechanisms triggered by GITR are poorly understood.
The data presented in this study indicate that GITR promotes proliferation, IL-2 production, and survival of naive T cells as well as costimulates previously activated T cells. Furthermore, GITR triggers the activation of three subsets of MAPKs and NF-B. However, these GITR-induced events are insufficient to directly inhibit AICD of primary T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BALB/c mice were purchased from The Jackson Laboratory. DO11.10 mice transgenic for a TCR specific for residues 323–339 of OVA (OVA323–339) were a kind gift from Dr. K. Murphy (Washington University School of Medicine, St. Louis, MO). All mice were kept at a specific pathogen-free facility in accordance with the guidelines of the animal studies committee of Washington University.
Antibodies
Polyclonal anti-IB
(BD Biosciences), monoclonal anti-actin (C4; Chemicon International), and HRP-conjugated polyclonal secondary Abs (Santa Cruz Biotechnology) were used for Western blot analysis. Abs specific for phosphorylated and total ERK, JNK, and p38 as well as MAPK-specific in vitro kinase kits were purchased from Cell Signaling Technology. Anti-CD3
(145-2C11), anti-CD4 (H129.1), anti-CD25 (PC61), anti-I-Ad (AMS-32.1), anti-B220 (RA3-6B2), anti-CD28 (PV.1), CTLA-4-Ig, anti-rat IgG1 (RG11/39.4), anti-rat IgG2a (RG7/1.30), and control rat IgG2a (R35-95) Abs were purchased from BD Biosciences. Anti-GITR-L Ab (YGL 386.2.2) was a kind gift of Dr. H. Waldmann (University of Oxford, Oxford, U.K.) and has been characterized previously (4). Anti-GITR Abs (BAF524 and 108619), control goat Ab, GITR-L, and anti-His mAb were purchased from R&D Systems. For flow cytometry, Abs were directly conjugated with FITC or PE as indicated or were biotinylated for detection with FITC-streptavidin (BD Biosciences).
Cell death assay
CD3-specific Ab was immobilized in 96-well plates at the indicated concentrations in PBS at 4°C overnight. Activated DO11.10 or BALB/c T cells (1.5 x 105/well) were stimulated with plate-bound anti-CD3 Ab and soluble anti-GITR or control Ab (5 µg/ml). After the times indicated in the figures, cell viability was determined by flow cytometry of 104 events on the basis of cell size and propidium iodide (PI) staining (0.25 µg/ml) or after staining with annexin V and PI according to the manufacturer’s protocol (Trevigen).
Cytokine production
DO11.10 splenocytes (2.5 x 106/well) were plated in 12-well plates with 0.1 µM OVA323–336 and the indicated Abs. Supernatants were harvested after 72 h and assayed for IL-2 levels using an ELISA kit (BD Biosciences).
Flow cytometric analysis (FACS) of surface expression of lymphocyte markers
Naive and in vitro activated lymphocytes (2.5 x 105 cells) were stained for 30 min at 4°C in 50 µl with the indicated Abs or appropriate isotype controls. The stained cells were washed three times and resuspended in PBS. Analysis was performed on gated live cells using FACScan or FACSCalibur flow cytometers (BD Biosciences) and WinMDI software (Joe Trotter).
In vitro activation of lymphocytes
Spleens were removed from DO11.10 or BALB/c mice and mechanically disrupted into a single cell suspension. Erythrocytes were removed by lysis in ACK buffer (150 mM KCl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.2). Five million lymphocytes per well were plated on six-well plates in lymphocyte medium (RPMI 1640 supplemented with 10% FBS, 4 mM L-glutamine, 10 mM HEPES, 100 mM nonessential amino acids, 57 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin). DO11.10 T cells were stimulated with 0.3 µM OVA323–339, whereas BALB/c T cells were activated by plate-bound CD3-specific (1 µg/ml) and soluble CD28-specific Abs (1 µg/ml) for 3 days. Activated lymphocytes were washed with PBS after primary stimulation and rested for 18 h in lymphocyte medium at 37°C. For subsequent analysis, live cells were isolated by centrifugation in Ficoll-Paque (Amersham Biosciences).
MAPK phosphorylation and in vitro kinase assays
Activated DO11.10 splenocytes (5 x 106 cells for detection of MAPK phosphorylation, 107 cells for in vitro kinase assay) were treated with GITR-specific or control Ab (5 µg/ml) at 37°C for the indicated times and handled according to the manufacturer’s specifications (Cell Signaling Technology), except that secondary Abs used to detect phosphorylated or total protein levels were obtained from Santa Cruz Biotechnology. Chemiluminescence was performed with reagents from Cell Signaling Technology or Amersham Biosciences.
Proliferation assays
Naive or activated cells (105) were plated in triplicate per condition in 96-well plates. When indicated, BALB/c splenocytes irradiated at 2000 rad were used as accessory cells. One microcurie per well of [3H]thymidine (ICN Biomedical) was added 8 h before the indicated time point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Studies of T cell lines transfected with GITR and GITR-deficient primary T cells activated by Con A suggested that the receptor modulates AICD (2, 3, 10). To define the molecular mechanism of GITR-induced inhibition of AICD under physiologic conditions, naive DO11.10 splenocytes were activated with Ag (OVA323–339) in vitro. Upon Ag stimulation, the vast majority of cells in the culture were activated CD4+ T cells that expressed high levels of GITR and CD25 (data not shown). Subsequently, activated lymphocytes were rested for 18 h before treatment with plate-bound Ab specific for CD3 to cause AICD. Analysis of cell size and PI staining by FACS revealed a significant decline in cell survival in response to CD3 stimulation (data not shown). In contrast to published studies that analyzed the effects of GITR overexpression, incubation of activated T cells expressing physiologic levels of GITR with GITR-specific Ab did not promote cell survival in comparison to control Ab, as indicated by similar percentages of PI– cells with a forward scatter typical for live cells in cultures 18 h post-restimulation. However, PI stains cells at a relatively late stage of apoptosis and does not distinguish between apoptotic and necrotic cells. To examine whether GITR stimulation affects earlier events, apoptosis was measured by double staining with annexin V and PI. Annexin V interacts with phosphatidylserine, which becomes exposed on the cell surface early during apoptosis while the cell membranes are still intact, i.e., the cells are PI–. Consistent with our previous data, incubation with GITR-specific Ab did not prevent early apoptotic events between 3 and 13 h post-restimulation of the cells with low or high concentrations of anti-CD3 Ab (Fig. 1A). At the 13 h time point, the apparent decrease in the percentage of early apoptotic events was the result of the increased percentage of late (annexin V+/PI+) apoptotic events (data not shown). To rule out that stimulation of T cells with GITR-specific Ab failed to block antiapoptotic events due to the rapid induction of proapoptotic pathways caused by CD3-specific Ab, activated T cells were incubated with anti-GITR or control Ab for 2 h before and continuously during restimulation by CD3 cross-linking. Consistent with our earlier results, this treatment was also insufficient to protect the T cells from AICD (Fig. 1B).
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GITR functions as a costimulatory receptor on T cells
GITR along with other members of the TNFR family, including Ox40, CD30, 4-1BB, and CD27, has been suggested to promote T cell activation similar to CD28 (12, 13). Hallmarks of costimulation include increased proliferation, cytokine production, and cell survival. To determine whether GITR costimulates activated T cells under conditions used to study AICD, activated DO11.10 T cells were treated with cognate Ag in the presence of GITR-specific or control Abs. GITR cross-linking using a specific Ab was sufficient to enhance Ag-dependent proliferation of previously activated T cells (Fig. 2A). Although the effect was less pronounced, GITR cross-linking also augmented T cell proliferation in the presence of exogenous APCs, which can costimulate via CD80/CD86 and other surface molecules. Furthermore, GITR-L expression was not detected on activated DO11.10 splenocytes, arguing that residual APCs, present as <4% B220+ cells and expressing adequate levels of I-Ad, provide suboptimal costimulation via GITR to T cells under conditions used to analyze AICD (data not shown). These results demonstrate that GITR can trigger costimulatory signals in previously activated T cells.
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Consistently, cross-linking of GITR with GITR-L or GITR-specific Ab also promoted the proliferation of splenocytes from naive BALB/c mice after treatment of cells with soluble or plate-bound CD3-specific Ab (Fig. 3A and data not shown). Reducing CD28 costimulation triggered by CD80/CD86 on APCs present in the cultures of nontransgenic T cells using CTLA-4-Ig did not interfere with the enhancement of proliferative responses due to GITR cross-linking with GITR-L or GITR-specific Ab (data not shown). CD28-specific Ab, which provided costimulation in the presence of CTLA-4-Ig, was included as a positive control in these studies. In contrast to activated T cells that were not protected against AICD by GITR cross-linking, naive T cells showed increased survival in response to GITR cross-linking, similar to CD28 cross-linking during primary stimulation (Fig. 3B). However, it is currently unclear whether this increased percentage of live cells is due to decreased apoptosis and/or proliferative expansion. These findings suggest that GITR plays a role in promoting cell survival of primary T cells during their initial stimulation, but is not sufficient to protect against AICD in the late phases of immune responses.
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B and MAPKs
To characterize signaling pathways that may contribute to GITR-induced cellular effects, we analyzed the activation of NF-B and MAPKs. NF-B is a critical regulator of cell survival and apoptosis, which is activated by a variety of TNFR family members, including GITR (4, 16). Dimeric NF-B molecules are sequestered in the cytoplasm by IBs, which are degraded in response to stimuli that cause NF-B activation and are resynthesized as NF-B-dependent gene products (17). To study NF-B activation under the conditions used to analyze AICD, IB levels were measured by Western blot of lysates of activated DO11.10 splenocytes that had been restimulated with GITR-specific or control Abs. Typical of TNFR-induced NF-B activation, IB levels initially decreased and later increased in primary T cells stimulated with anti-GITR Ab compared with the negative controls (Fig. 4 and data not shown). Combined with the analyses of cell viability of activated T cells presented above, these results indicate that the net effect of GITR-induced NF-B activation neither promotes nor inhibits AICD.
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B, MAPKs play crucial roles in regulating cell survival and apoptosis and can be activated by a variety of TNFR family members (16, 18). In response to GITR-induced signaling in primary T cells, we monitored MAPK activation by Western blot using Abs specific for phosphorylated forms of the three MAPK families. After treatment of activated DO11.10 splenocytes with GITR-specific Ab, activation of ERKs, JNKs, and p38 was increased, and peak phosphorylation levels were observed 20 min post-GITR cross-linking (Fig. 5A). JNK2 was the predominant JNK protein expressed and phosphorylated in DO11.10 lymphocytes, whereas only minute expression and phosphorylation of JNK1 were detected. Although ERK1 expression was substantially lower than ERK2 expression, the phosphorylation of both MAPKs reached similar magnitudes. However, ERK2 activation occurred much earlier and was more prolonged than ERK1 activation. Similar to ERK2, phosphorylation of p38 was also evident at 1 min poststimulation and was still present at 1 h poststimulation. To verify that phosphorylation of MAPKs correlates with increased kinase activity, in vitro kinase assays for p38, JNKs, and ERKs were performed. Compared with control Ab, GITR-specific Ab induced phosphorylation of the p38 substrate ATF2 (Fig. 5B). Similar results were obtained with the respective substrates of JNK and ERK, c-Jun and Elk1 (data not shown). Taken together, our results indicate that GITR can trigger the activation of all three MAPK subfamilies. However, similar to NF-B activation and other costimulatory events triggered by GITR-induced signaling pathways, the overall impact of increased MAPK activation was insufficient to affect AICD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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TNFR-related proteins lacking a death domain have been implicated in pathways promoting cell survival via activation of NF-B (16). Consistently, GITR is thought to inhibit cell death pathways in lymphocytes, and GITR-deficient T cells have been shown to be more sensitive to AICD, a form of apoptosis that controls lymphocyte homeostasis (2, 3, 10). However, it is unclear whether GITR activates antiapoptotic signaling pathways directly or affects cell survival indirectly via the production of cytokines such as IL-2 and receptors such as IL-2R and Fas that control pro- and antiapoptotic pathways. To test the role of GITR-induced signaling events in AICD, activated T cells were restimulated with CD3-specific Ab and either GITR-specific or control Ab. In contrast to studies of cells deficient in or transfected with GITR, cross-linking of endogenous GITR did not protect activated primary lymphocytes from AICD (Fig. 1). Consistent with previously published findings, GITR cross-linking with specific Ab or engagement by its ligand did result in increased proliferation, IL-2 production, and survival of T cells during primary stimulation as well as augmented the proliferation of activated T cells (Figs. 2 and 3). No differences in the rate of AICD were detected despite activation of the canonical NF-B pathway and MAPKs (Figs. 4 and 5). Therefore, the experimental conditions used to study the effects of GITR-induced events were sufficient to trigger signaling pathways and cellular functions in T cells. Importantly, these findings indicate that the contribution of GITR to cell survival pathways depends upon the stage of T cell activation and stimulus used to induce cell death.
Our data suggest that the effects of GITR-induced signal transduction are regulated by additional signals that result in differences in the survival of naive and activated T cells. Wajant (19) discusses how NF-B activation leads to coordinated expression of prosurvival proteins, including members of the Bcl-2 family and proapoptotic gene products, such as TRAIL. By establishing the balance between cell survival and apoptosis through changes in gene expression, NF-B promotes cellular expansion during the acute phase of an immune response, yet provides regulatory mechanisms to control inflammation. Beyond controlling NF-B activity, GITR has been shown to interact with the proapoptotic protein Siva, which may initiate cell death pathways and could further counterbalance the cytoprotective effects of NF-B (8). As mentioned by Riccardi et al. (20), adapter proteins that interact with GITR could potentially regulate cellular events triggered by the receptor, such as regulation of T cell survival. Recently, we have shown that TNFR-associated factor 2 (TRAF2) interacts with GITR in T cells and functions as a negative regulator of NF-B activation induced by the receptor (21). Moreover, TRAF2 can regulate the production of reactive oxygen species induced by TNFR-related proteins, which can shift the balance between cell survival and death (22). Because TRAF2 is a central regulator of cell survival and apoptotic pathways triggered by TNFR-related proteins, it is intriguing to speculate that TRAF2-mediated signaling is involved in deciding the ultimate fate of T cells that receive stimulation through GITR (23). These possibilities and whether GITR plays distinct roles in effector T cells and Treg cells need to be further addressed to establish the role of GITR in T cell survival.
Distinct MAPK family members, such as p38 and JNK, can be activated by TNFR-related proteins and regulate lymphocyte functions, survival, and apoptosis (16, 24). Western blot analysis and in vitro kinase assays indicated that GITR can induce activation of the MAPK subfamilies, JNK, ERK, and p38 (Fig. 5). Although quantitative differences in phosphorylation were detected due to dissimilar expression levels of the distinct MAPKs in lymphocytes, activation of all three kinase subfamilies peaked 20 min after stimulation by GITR cross-linking. These results suggest a role for MAPKs in regulating the GITR-induced responses of effector T cells and/or Treg cells.
In summary, GITR promotes the activation, survival, and cytokine production of T cells. Furthermore, GITR can induce the activation of NF-B and three subfamilies of MAPKs, ERKs, JNKs, and p38. However, costimulatory signaling events triggered by GITR do not directly inhibit AICD. Understanding the molecular details of cell survival pathways triggered by GITR in T cells will provide further insights into the mechanisms involved in physiologic and pathologic immune responses.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported in part by grants from the National Institutes of Health (5RO1HL67312-02 and T32HL007317). 
2 Address correspondence and reprint requests to Dr. Robert H. Arch, Washington University School of Medicine, Campus Box 8052, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: arch{at}wustl.edu
3 Abbreviations used in this paper: GITR, glucocorticoid-induced TNFR; AICD, activation-induced cell death; GITR-L, GITR ligand; PI, propidium iodide; TRAF, TNFR-associated factor; Treg, regulatory T cell.
Received for publication August 10, 2004. Accepted for publication April 5, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B at the crossroads of life and death. Nat. Immunol. 3: 221-227.[Medline]
B signaling: a complex relationship. Vitam. Horm. 67: 101-132.[Medline]
B activation. J. Immunol. 174: 7875-7882.B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7: 715-725.[Medline]
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