CD8 T Cell-Intrinsic GITR Is Required for T Cell Clonal Expansion and Mouse Survival following Severe Influenza Infection

Laura M. Snell, Ann J. McPherson, Gloria H. Y. Lin, Shimon Sakaguchi, Pier Paolo Pandolfi, Carlo Riccardi and Tania H. Watts
J Immunol December 15, 2010, 185 (12) 7223-7234; DOI: https://doi.org/10.4049/jimmunol.1001912

Abstract

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-xL. In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphoctye survival, they clearly have nonredundant functions in protection from severe infection.

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNFR superfamily that modulates immune responses. Perhaps most extensively studied for its high basal expression on CD4+CD25+Foxp3+ regulatory T cells (Tregs) (1, 2), GITR is also expressed on many other immune cell types, including CD4 and CD8 T cells, NK cells, B cells, and macrophages (3). The ligand for GITR (GITRL) can be expressed on dendritic cells, macrophages, B cells, endothelial cells, and activated T cells (3).

The role of GITR in immune responses is complex and may be cell-type specific. Stimulation of macrophages with an anti-GITR Ab induces a proinflammatory response (4); however, recent reports have shown that GITR can play an inhibitory role for human NK cell activation (5, 6). GITR has also been extensively studied in the context of Tregs and generated much excitement when it was observed that agonistic anti-GITR Ab in cocultures of CD4+CD25− effector T cells and CD4+CD25+ Tregs caused the abrogation of suppression by Tregs (1, 2). Interestingly, it was shown that GITR signaling on the effector T cells was necessary to render the cells less refractory to Treg-mediated suppression (7). A recent report showed that this phenomenon is not unique to CD4 T cells, and GITR signaling can induce resistance to CD4+CD25+ Treg suppression in CD8 T cells as well (8).

In addition to enhancing T cell responses by raising the threshold for Treg suppression, GITR has also been shown to costimulate CD4 and CD8 T cells in vitro by enhancing proliferation, survival, and cytokine secretion (9, 10). GITR acts early in the immune response to lower the threshold for CD28 signaling in both CD4 and CD8 T cells (11). Whereas GITR’s costimulatory role for T cells is well documented in vitro, much remains to be elucidated as to its function in vivo, particularly on CD8 T cells. Notably lacking from the literature is any thorough assessment of GITR’s physiological role on CD8 T cells in an infectious disease context. Although a few studies have shown that agonistic anti-GITR Ab can enhance antiviral CD8 T cell responses in vivo (12–14), the mechanism whereby this occurs remains to be determined, as GITR is broadly expressed and its signaling on other immune cells may influence the CD8 T cell response indirectly. Clearly, more defined models are needed to study the intrinsic role of GITR on CD8 T cells in response to pathogens in vivo.

In this study, we sought to address the role of GITR on CD8 T cells during viral infection. By using an adoptive transfer model in which GITR is only absent on the responding CD8 T cells, we show that GITR is intrinsically required on CD8 T cells for maximal primary and secondary CD8 T cell responses to influenza. Moreover, we demonstrate that GITR-specific agonistic Abs enhance the response to influenza virus through direct effects of GITR on CD8 T cells. Although GITR does not influence cell division, it augments CD8 T cell survival by upregulating the prosurvival molecule Bcl-xL in a NF-κB–dependent manner. We found that GITR-induced NF-κB activation is positively regulated by both TNFR-associated factor (TRAF) 2 and 5, whereas TRAF1 is dispensable for this survival signaling. Although the absence of GITR on T cells did not affect disease outcome during mild influenza virus infection, during a severe and potentially lethal model of influenza infection we show that GITR on CD8 T cells augmented viral clearance and protected mice from death. These results highlight the importance of GITR as a CD8 T cell-intrinsic costimulatory receptor.

Materials and Methods

Mice

C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). CD45.1 and Thy1.1 congenic mice were crossed with OT-I mice (The Jackson Laboratory, Bar Harbor, ME). GITR−/− mice have been previously described (15). GITR−/− mice were backcrossed onto the C57BL/6 background for at least eight generations and then further crossed to generate CD45.1 GITR−/− OT-I mice. TRAF1−/− mice (16) were originally provided by E. Tsitisikov (Center for Blood Research, Boston, MA), and crossed to OT-I TCR transgenic mice, as previously reported (17). Mice were maintained under specific pathogen-free conditions in sterile microisolator cages. Animal studies were approved by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on Animal Care.

Influenza virus infection

Six- to 10-wk-old C57BL/6 and GITR−/− mice were infected intranasally (i.n.) with 5 hemagglutinin units (HAU) influenza A/HK-X31 (H3N2). Thirty days postinfection, some mice were challenged with 5 HAU influenza A/PR8 (H1N1). For survival experiments using influenza A/PR8, mice were infected i.n. with 105 tissue culture infectious dose 50 (TCID50)/mouse. For immunization after adoptive transfer of wild-type (WT)/GITR−/− OT-I T cells, mice were infected with 5 HAU or 6118 TCID50/mouse A/HK-X31-OVA (X31-OVA) or 106 TCID50/mouse A/PR8-OVA (PR8-OVA) (18), both generously provided by P. Doherty and P. Thomas (St. Jude Children’s Research Hospital, Memphis, TN). At the indicated times after X31-OVA infection, spleen, mediastinal lymph node, bone marrow, and lungs were harvested. Lungs were perfused with PBS, and lymphocytes were enriched by isolation over an 80/40% Percoll gradient. Single-cell suspensions were prepared from all organs and were subjected to flow cytometry staining. After PR8-OVA or PR8 infection, mice were sacrificed when they had lost 30% body weight or were moribund.

Flow cytometry

MHC class I tetramers were obtained from the National Institute for Allergy and Infectious Diseases tetramer facility (Emory University, Atlanta, GA). Influenza nucleoprotein (NP)366–374-specific CD8 T cells were surface stained with anti-CD8α, anti-CD62L (eBioscience, San Diego, CA), and Db/NP366–374 tetramers. For intracellular IFN-γ staining, splenocytes were restimulated with 1 μM NP366–374 for 6 h with Golgi Stop (BD Biosciences, San Jose, CA) at 37°C. Cells were surface stained as above, fixed, and intracellularly stained for IFN-γ (BD Biosciences). Adoptively transferred OT-I cells were detected using anti-Thy1.1 and anti-CD45.1 (eBioscience) in conjunction with anti-CD8α. For intracellular IFN-γ staining, splenocytes and lymphocytes isolated from lung were restimulated at 37°C with 1 μM OVA257–64 using Golgi Stop for 6 and 4 h, respectively. Cells were then surface stained for CD8, CD45.1, and/or Thy1.1, fixed, and intracellularly stained for IFN-γ (BD Biosciences). For detection of degranulation, 5 μg/ml anti-CD107a (BD Biosciences) was added at the beginning of the restimulation culture. Fluorescent minus one controls or unstimulated samples (no peptide) were used as negative controls. Certain experiments also required staining with anti-GITR, anti-CD69, anti-CD44, anti-CD4, and anti-CD25 (eBioscience). Foxp3 staining was performed using a mouse Treg staining kit (eBioscience). Staining for apoptosis was carried out by adding annexin V (BD Biosciences) to surface-stained cells. Samples were analyzed using a FACSCalibur (BD Biosciences) and FlowJo (Tree Star, Ashland, OR) software.

T cell isolation, adoptive transfers, and agonistic Ab studies

WT and GITR−/− OT-I T cells were purified from lymph nodes and spleens of naive mice using a negative selection mouse CD8 T cell enrichment kit (StemCell Technologies, Vancouver, Canada). For memory experiments, memory-like WT and GITR−/− OT-I cells were generated, as previously described (19). In certain experiments, T cells were stained with 1 μM CFSE for 10 min at 37°C. T cells were injected i.v. at 104 cells/mouse (for ratio experiments 5 × 103 of each cell type/mouse) with the exception of CFSE experiments, which used 106 cells/mouse, and PR8-OVA experiments, which used 103 cells/mouse. A day later, the mice were infected with X31-OVA or PR8-OVA, as described above. For experiments using agonistic Abs, X31-OVA was administered i.p. at a dose of 100 HAU/mouse with either 200 μg anti-GITR (DTA-1) hybridoma (2) or purified rat IgG (Sigma-Aldrich, St. Louis, MO).

Cell viability assay

WT and GITR−/− OT-I CD8 T cells were isolated from naive mice and cultured in vitro with irradiated splenocytes from C57BL/6 mice at a ratio of 1:4. Cultures were stimulated with a suboptimal dose of SIINFEKL (10−10 M), and in some cases T cells were CFSE labeled, as described above. CFSE analysis was performed on day 2, and annexin V staining of cultures was performed on days 0, 2, and 3.

Signaling studies: small interfering RNA knockdowns

OT-I T cells and, in some experiments, TRAF1−/− OT-I cells were stimulated in vitro at 0.1 μg/ml SIINFEKL. Cells were subjected to lympholyte (Cedarlane Laboratories, Hornby, Ontario, Canada) on day 3 of the culture, rested, and then stimulated with either 10 μg/ml DTA-1 or rat IgG. In some experiments, cells were preincubated with either 20 μg/ml NF-κB inhibitor 1-pyrrolidinecarbodithioic acid [PDTC], ammonium salt (Calbiochem, San Diego, CA), or vehicle for 1 h prior to DTA-1 or rat IgG stimulation. For small interfering RNA (siRNA) knockdown experiments, cells were cultured with 20 ng/ml IL-15 (R&D Systems, Minneapolis, MN) for 72 h after lympholyting. A total of 9 × 106 OT-I cells was then transfected with 1 μM siRNA targeting TRAF2, TRAF5, both TRAF2 and TRAF5, or a control scrambled duplex RNA (IDT Technologies, Coralville, IA). Transfections were performed using the Amaxa mouse T cell Nucleofector kit (Lonza, Cologne, Germany). Cells were rested for 20 h and then stimulated with 10 μg/ml of either DTA-1 or control rat IgG.

Western blots

Cells were lysed in 1% Nonidet P-40 with complete protease inhibitor mix (Roche, Basel, Switzerland). Lysates were quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA). Lysates were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were probed with Abs specific for IκBα, Bcl-xL, TRAF2 (Cell Signaling Technology, Beverly, MA), TRAF5 (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma-Aldrich), and incubated with HRP-conjugated anti-rabbit or anti-goat IgG (Sigma-Aldrich, Oakville, Ontario, Canada). Signals were revealed by chemiluminescence (GE Healthcare, Baie D'Urfe, Quebec, Canada) and visualized by autoradiography. Where indicated, quantitation was performed using Quantity One software (Bio-Rad, Hercules, CA).

Viral clearance

Lungs were excised from mice at various time points after X31-OVA and PR8-OVA infection and then homogenized in RPMI 1640 medium (1 g lung tissue/10 ml). Supernatant was obtained and stored at −70°C. Viral loads were determined by the Madin-Darby canine kidney assay with the Reed and Muench technique, as previously described (20).

Statistical analysis

Where indicated, p values were obtained using the Student t test, unpaired or paired for ratio experiments (two tailed, 95% confidence interval). The log-rank test was used to determine significance for mouse survival experiments. Statistically significant differences are indicated as *p < 0.05, **p < 0.01, or ***p < 0.001.

Results

GITR on the T cells is essential for maximal CD8 T cell responses to influenza virus

To examine the role of GITR on CD8 T cells during viral infection in vivo, we i.n. infected GITR−/− and WT C57BL/6 mice with influenza A/HK-X31 (H3N2) virus. Some mice were challenged with the serologically distinct influenza A/PR8 (H1N1) 30 d after the initial X31 infection to examine the secondary response (Supplemental Fig. 1A). Ag-specific CD8 T cell responses to the immunodominant NP366–374 epitope were assessed using fluorescently labeled H-2Db/NP366–374 tetramers. Decreased influenza-specific CD8 T cell numbers were observed in GITR−/− mice in a subset of organs, but only at particular times of the immune response (Supplemental Fig. 1B–F). As GITR can be expressed on many different cell types during the course of an infection (3), it was possible that the lack of GITR signaling on other cell types in the local environments, such as Tregs, was indirectly affecting the CD8 T cell response.

Therefore, to dissect the CD8 T cell-intrinsic effects of GITR during viral infection in vivo, we crossed GITR−/− mice with CD45.1 OT-I mice, whose CD8 T cells have transgenic TCRs specific for the H-2Kb/OVA257–264 epitope. GITR−/− mice had been backcrossed at least eight times before a further backcross to generate the GITR−/− OT-I transgenic mice. Purified CD8 T cells from CD45.1 GITR−/− OT-I and Thy1.1 WT OT-I mice were mixed at a ratio of 1:1 and adoptively transferred into WT CD45.2 recipients (Fig. 1A, 1B). One day later, mice were infected with influenza A/HK-X31-OVA (X31-OVA), which carries the OVA257–264 epitope in its neuraminidase stalk (18). The ratio of WT to GITR−/− OT-I T cells at the peak of the response was >1 in all organs assayed, indicating significant defects in the recovery of GITR−/− OT-I T cells in spleen, lung, mediastinal lymph node, and bone marrow (Fig. 1C, 1D). Differences ranged from 2- to 4-fold, depending on the particular organ. A similar defect in GITR−/− T cell recovery was observed at days 7, 9, and 12 postinfection (Fig. 1E). Thus, the defect in recovery of GITR-deficient CD8 T cells could not be attributed to a difference in the kinetics of expansion between WT and GITR−/− OT-I T cells. Therefore, these results demonstrate that GITR is required on the CD8 T cells for their maximal expansion during the primary response to influenza virus.

FIGURE 1.
FIGURE 1.

Analysis of CD8 T cell-intrinsic effects of GITR during influenza infection. A, Thy1.1 OT-I and CD45.1 GITR−/− OT-I CD8 T cells (5 ×103 of each) were injected as a 1:1 mixture into WT mice. One day later, the mice were infected i.n. with X31-OVA. On days 7, 9, and 12, the mice were sacrificed, and the ratio of WT/GITR−/− OT-I T cells was analyzed. Data are representative of two independent experiments, each using four to five mice. B, Representative FACS plot of original 1:1 Thy1.1 OT-I/CD45.1 GITR−/− OT-I ratio upon injection. Plot gated on CD8 T cells. C, Ratio of WT/GITR−/− OT-I T cells in various organs at day 9. D, Representative FACS plots of Thy1.1 and CD45.1 gating for ratio determination. E, Kinetics of WT and GITR−/− OT-I primary response. Graphs show percentage of transferred cells of total CD8 T cells at days 7, 9, and 12. At each time point, five mice were used. F and G, Percentage of IFN-γ–positive (F) and percentage of CD107a-positive cells (G) of transferred Thy1.1 OT-I or CD45.1 GITR−/− OT-I T cells. Representative FACS plots of data in F and G are shown on the left. Plots are gated on CD8 T cells and then either Thy1.1 or CD45.1 cells. No Ag, unstimulated controls.

Competitive models can sometimes exacerbate differences between two cell populations, as each population is forced to compete for the same pool of resources. As the previous experiments were performed in a competitive fashion, we also investigated whether GITR−/− OT-I cells would show a similar impairment when they did not have to compete with OT-I cells for survival factors. When CD45.1 OT-I or CD45.1 GITR−/− OT-I T cells were injected into separate mice and infected with X31-OVA, similar defects were observed, although slightly delayed, particularly in the lung (Supplemental Fig. 2).

Although the GITR−/− OT-I mice had been extensively backcrossed (n = 9), to rule out any differences in cell recovery due to rejection, we injected a 1:1 mix of in vitro expanded (see Materials and Methods and below) memory-like WT and GITR−/− OT-I T cells (3 million cells total) into mice and monitored their persistence without infection. At day 21 postinjection, both cell types could still be detected, and the GITR−/− T cell numbers were similar and, if anything, slightly higher in number than their WT counterparts (data not shown), indicating that the defects observed at day 7–12 of the primary response could not be attributed to rejection of the knockout cells.

GITR regulates CD8 T cell numbers, but not effector function

Multiple costimulatory molecules in the TNFR superfamily have been implicated in enhancing not only the magnitude of the T cell response, but also its quality (21, 22). To assess whether GITR was also regulating effector function of the responding CD8 T cells, we examined IFN-γ production and the expression of CD107a, a marker of degranulation, on WT and GITR−/− OT-I cells at the peak of the primary response (Fig. 1F, 1G). Despite the fact that there were fewer GITR−/− OT-I T cells overall, the proportion of GITR−/− cells that was producing IFN-γ and expressing CD107a was similar to the proportion of WT OT-I cells showing this phenotype. Thus, whereas GITR is critical for maximal CD8 T cell expansion in the primary response to influenza, it does not influence effector function per T cell.

GITR on the T cells is essential for their secondary expansion in response to influenza virus

Under some circumstances, memory T cells are less dependent on costimulation than their naive counterparts (23). Therefore, we next assessed whether GITR would play a role in the recall CD8 T cell response to influenza virus. As there were already significant defects in CD8 T cell numbers after the primary response, to avoid the complications of these defects and focus on the effect of GITR during the secondary response, we generated memory-like cells in vitro, by culturing WT and GITR−/− OT-I splenocytes with Ag followed by IL-15 treatment (19). At the end of the cultures, the WT and GITR−/− OT-I cells showed a similar memory phenotype: CD44high, CD69low, with GITR expression detected on the WT cells (Fig. 2B). A 1:1 mixture of Thy1.1 OT-I and CD45.1 GITR−/− OT-I memory-like cells was adoptively transferred into WT recipient mice and, a day later, the mice were challenged with X31-OVA (Fig. 2A). At several time points throughout the response, the number of WT OT-I cells consistently outnumbered the number of GITR−/− OT-I cells in all organs examined (Fig. 2C, 2D). As in the primary response, the proportion of WT and GITR−/− cells producing IFN-γ and expressing CD107a after ex vivo stimulation was approximately equal (Fig. 2E, 2F). GITR, therefore, is important for the maximal expansion of CD8 T cells in both the primary and recall response to influenza virus, and this effect is intrinsic to the CD8 cells.

FIGURE 2.
FIGURE 2.

Role of CD8 T cell-intrinsic effects of GITR in recall responses of OT-I memory T cells in vivo. A, Thy1.1 OT-I and CD45.1 GITR−/− OT-I memory-like T cells were generated in vitro and were injected as a 1:1 mixture into WT mice as in Fig. 1. A day later, the mice were infected i.n. with X31-OVA. On days 6, 8, and 10, the mice were sacrificed, and the ratio of WT/GITR−/− OT-I T cells was analyzed. Data are representative of two independent experiments, each using four to five mice. B, GITR, CD69, and CD44 expression on naive, activated, and memory T cells was analyzed at days 0, 2, and 9, respectively, of in vitro memory cell culture. C, Ratio of WT/GITR−/− OT-I memory-like T cells in various organs at day 8. D, Kinetics of WT and GITR−/− OT-I secondary response. Graphs show percentage of transferred cells of total CD8 T cells at days 6, 8, and 10 in various organs. At each time point, four mice were used. E, Percentage of IFN-γ–positive cells of transferred Thy1.1 OT-I or CD45.1 GITR−/− OT-I T cells in spleen and lung. F, Percentage of CD107a-positive cells of transferred Thy1.1 OT-I or CD45.1 GITR−/− OT-I T cells in spleen.

Agonistic anti-GITR Ab enhances effector T cell expansion through direct effects on the CD8 T cells

The agonistic anti-GITR Ab DTA-1 enhances effector T cell responses in vivo (3). DTA-1 was originally thought to mediate its effects by directly inhibiting Treg suppression (1, 2). However, this has recently come under question (7, 24), as GITR can also costimulate T cells and make them refractory to Treg suppression (7, 9). To test the CD8 T cell-intrinsic role of GITR in response to the anti-GITR agonist, we compared the response of WT and GITR−/− OT-I T cells to X31-OVA in the presence of DTA-1 or control rat IgG Ab (Fig. 3A). For this experiment, we used the i.p. route of infection, which results in minimal virus replication. At the dose of virus chosen, with this route of infection, the response of the OT-I T cells to the virus is very weak and almost completely dependent on inclusion of the DTA-1 agonist, focusing the analysis in this experiment on the DTA-1–dependent effects. At the peak of the primary response, there was a 3-fold increase in the proportion of OT-I T cells recovered after the administration of DTA-1 as compared with rat IgG (Fig. 3B). Strikingly, this effect was lost if the T cells lacked GITR (Fig. 3B). Thus, DTA-1 enhances CD8 T cell expansion largely through direct effects on the CD8 T cells.

FIGURE 3.
FIGURE 3.

GITR is required on the CD8 T cells for enhancement of CD8 T cell expansion by anti-GITR in vivo. A, A total of 104 CD45.1 OT-I or CD45.1 GITR−/− OT-I T cells was transferred into WT mice. One day later, the mice were infected i.p. with 100 HAU/mouse X31-OVA and 200 μg/mouse of DTA-1 or rat IgG. Mice were sacrificed at day 7. Data were pooled from two independent experiments. B, Percentage (middle panel) and number (right column) of CD45.1 cells of total CD8 T cells in spleen. Representative FACS plot of data in B is shown on the left. Plots are gated on CD8 T cells. C, Percentage (middle column) and number (right column) of CD44high cells of CD4 T cells in spleen. Representative FACS plot of data in C is shown on the left. Plots are gated on CD4 T cells. D, Percentage (middle column) and number (right column) of Foxp3+CD25+ of CD4 T cells in spleen. Representative FACS plot of data in D is shown at left. Plots are gated on CD4 T cells.

Although our model did not allow us to specifically examine CD4 T cell-intrinsic effects of DTA-1, we did observe a modest enhancement in the number of activated CD4 T cells, defined as having a CD44high phenotype, in the mice that had been administered DTA-1 versus rat IgG (Fig. 3C). In addition, it has been reported that DTA-1 can also costimulate Tregs, causing them to proliferate (1, 9, 24, 25). We surveyed the number of CD4+CD25+Foxp3+ cells in the mice and found that there was a trend toward slightly higher numbers of Tregs with DTA-1 treatment (Fig. 3D). Whereas DTA-1 can costimulate both CD4 effectors and Tregs in our model, the increase in CD8 T cell expansion observed with DTA-1 treatment was found to be almost exclusively through CD8 T cell-intrinsic effects.

Role of GITR in the survival of CD8 T cells responding to influenza infection

Having demonstrated that GITR is essential for maximal CD8 T cell responses to influenza infection, we next sought to dissect the mechanism by which GITR was regulating cell recovery. The defect in numbers of GITR−/− OT-I cells in both the primary and recall responses of the i.n. influenza model could be attributable to differences in homing or a defect in either proliferation or survival. To study homing, the seeding of the cells was examined prior to influenza infection, 24 h after a 1:1 mixture of WT:GITR−/− OT-I cells was injected into mice. The ratio in all organs examined was close to 1 (data not shown), implying that the cells homed in an equal manner, at least before they encountered Ag. Moreover, the finding that defects in cell recovery of GITR−/− CD8 T cells were observed in all organs examined, and that kinetic studies in the lung showed equal numbers of both WT and GITR−/− OT-I T cells early, with defects accumulating only later, argues against a trafficking defect.

To investigate whether the defect in recovery of GITR−/− OT-I T cells was due to a proliferative difference, WT mice were injected with CFSE-labeled WT or GITR−/− OT-I cells and infected with X31-OVA. We observed that both the number of divisions and the proportion of WT and GITR−/− OT-I cells that had undergone cell division were very similar (Fig. 4A). These results suggest that GITR is dispensable for Ag-dependent CD8 T cell division in vivo.

FIGURE 4.
FIGURE 4.

GITR on CD8 T cells is dispensable for proliferation, but required for CD8 T cell survival. A, A total of 106 CFSE-labeled CD45.1 OT-I or CD45.1 GITR−/− OT-I T cells was transferred into WT mice. One day later, mice were infected with X31-OVA and 3 d postinfection were sacrificed and CFSE analyzed via flow cytometry. Shown are representative FACS plots of CD45.1 gating and CFSE staining of transferred cells. Four to five mice were used per group in three independent experiments. B–D, Purified CFSE-labeled WT and GITR−/− OT-I T cells were cultured with irradiated splenocytes and 10−10 M SIINFEKL. B, CFSE analysis of WT and GITR−/− OT-I cells at day 2 of culture. C, Annexin V analysis on days 2 and 3. Plots are gated on CD8 CD45.1 T cells. D, Percentage of annexin V-positive cells of CD45.1 cells on days 2 and 3. Data in all panels are representative of two to four independent experiments.

It is difficult to examine cell death in vivo because apoptotic cells are cleared very quickly by macrophages (26). Therefore, to test whether GITR−/− OT-I cells had a survival defect, we moved to an in vitro model. Equal numbers of either WT or GITR−/− OT-I CD8 T cells were cultured with irradiated B6 splenocytes and a suboptimal dose of peptide (10−10 M). At the beginning of the culture, the percentage of annexin V and propidium iodide-positive WT and GITR−/− OT-I cells was approximately equal (data not shown). By day 2, the cells had begun to proliferate and CFSE labeling indicated that approximately equal numbers of WT and GITR−/− OT-I cells had undergone division (Fig. 4B), consistent with the in vivo data. Whereas the proliferation of the two populations was similar, there was a marked difference in the survival of WT versus GITR−/− OT-I cells. On day 2, a higher proportion of GITR−/− OT-I cells compared with OT-I cells was annexin V positive and primed to undergo apoptosis (Fig. 4C, 4D). This trend became statistically significant by day 3, as the GITR−/− OT-I cells became increasingly annexin V positive. Interestingly, at a peptide dose that was too low to induce proliferation (10−12 M), both WT and GITR−/− OT-I cells had the same percentage of annexin V-positive cells (data not shown). This result implies that the difference in survival of WT and GITR−/− cells is TCR signal dependent and not due to some intrinsic homeostatic difference between the WT and GITR−/− OT-I cells. Based on these data, we infer that GITR signaling on CD8 T cells is required for their in vivo survival.

GITR enables survival signaling via enhanced Bcl-xL expression downstream of NF-κB

Activation of the NF-κB signaling pathway is essential for cell survival. It was previously reported that GITR can initiate NF-κB signaling in primary T cells (11, 25, 27). To confirm this in our model, OT-I CD8 T cells were activated with peptide and subsequently stimulated with anti-GITR (DTA-1) or rat IgG. Activating the cells first and then stimulating with DTA-1 allows GITR to be upregulated above basal levels before its triggering, mimicking the situation that would occur during an Ag-specific immune response in vivo. Stimulating activated CD8 T cells with DTA-1 led to NF-κB activation as observed by IκBα degradation (Fig. 5A). In contrast, stimulation with control rat IgG did not lead to significant IκBα degradation.

FIGURE 5.
FIGURE 5.

GITR induces Bcl-xL by a NF-κB–dependent signaling pathway involving TRAF2 and TRAF5. A, Activated OT-I T cells were stimulated with anti-GITR (DTA-1) or rat IgG and analyzed for IκBα degradation (A) or Bcl-xL expression (B). C, Activated OT-I cells were preincubated with 20 μg NF-κB inhibitor, PDTC, or vehicle for 1 h. Cells were then stimulated with DTA-1 or rat IgG and analyzed for Bcl-xL expression 3 h poststimulation. D, WT OT-I T cells were activated in vitro, cultured with IL-15, and transfected with siRNA targeting T2, T5, both, or a scrambled control. Representative blots of TRAF2 and TRAF5 siRNA knockdowns. E, siRNA-treated cells were stimulated with either DTA-1 or rat IgG and analyzed for IκBα degradation. Representative IκBα blot at 30 min poststimulation. F, Graphs show DTA-1/rat IgG IκBα ratio at 15 and 30 min. Data are pooled from two independent experiments. KD, knockdowns; Scr, scrambled; T2, TNFR-associated factor 2; T5, TNFR-associated factor 5.

To further dissect the mechanism by which GITR enables survival signaling, we examined the expression of Bcl-xL, a prosurvival molecule downstream of NF-κB (28). When activated OT-I cells were stimulated with DTA-1, Bcl-xL expression was significantly enhanced compared with rat IgG stimulation, as measured at 3 h and as late as 20 h poststimulation (Fig. 5B). In contrast, when the OT-I cells were preincubated with a NF-κB inhibitor, PDTC, DTA-1 stimulation could no longer induce Bcl-xL to levels greater than in the rat IgG control, as measured at 3 h poststimulation (Fig. 5C). Similar results were seen at 20 h (data not shown); however, by this time the cells treated with the NF-κB inhibitor were beginning to die, presumably from the inhibition of this critical pathway. These results suggest that GITR regulates CD8 T cell survival, at least in part, by upregulating the expression of the prosurvival molecule Bcl-xL downstream of NF-κB signaling.

TRAF2 and TRAF5, but not TRAF1, enhance GITR-induced NF-κB survival signaling

Signaling downstream of TNFR family members is known to be mediated by the recruitment of TRAFs to the activated receptor (29). To further understand how GITR induces survival signaling, we investigated the role of several TRAF family members in mediating activation of NF-κB downstream of GITR. GITR has been shown to bind TRAFs 1, 2, and 3 via yeast–2-hybrid and overexpression studies (30, 31). In addition, TRAF5 has been shown to enhance NF-κB signaling downstream of GITR (32). The role of TRAF2 in GITR-mediated NF-κB signaling remains controversial (30, 31), and TRAF1, which is known to modulate TRAF2 signaling (33–36), has not been well investigated downstream of GITR. We sought, therefore, to determine the role of TRAFs 1, 2, and 5 in the activation of NF-κB downstream of GITR in CD8 T cells. To do this, we made use of siRNA to knock down TRAF2 and TRAF5, as well as use of TRAF1−/− OT-I mice. OT-I cells were activated in vitro and transfected with siRNA targeting TRAF2, 5, both TRAF2 and 5, or a scrambled siRNA control (Fig. 5D). NF-κB activation was diminished in individual TRAF2 and TRAF5 knockdown cells treated with DTA-1, as observed by decreased degradation of IκBα compared with the scrambled control (Fig. 5E, 5F). In cells in which both TRAF2 and 5 were knocked down, an additive effect was observed, with a higher level of intact IκBα persisting compared with the individual TRAF knockdowns (Fig. 5F). In contrast, when preactivated TRAF1−/− OT-I T cells were stimulated with DTA-1, the lack of TRAF1 did not significantly affect GITR-induced NF-κB signaling (Supplemental Fig. 3). Thus, TRAF1 is dispensable for GITR-induced NF-κB signaling, whereas TRAF2 and TRAF5 act as positive regulators to enhance NF-κB signaling downstream of GITR.

GITR protects against death following severe influenza infection

Although GITR was required for maximal T cell expansion during mild influenza infection (Fig. 1), it had no impact on mouse survival (data not shown). Therefore, to examine the potential role of GITR on influenza disease outcome, we turned to a more severe respiratory infection model using influenza A/PR8. Following i.n. infection with influenza A/PR8 (Fig. 6A), only 25% of the GITR−/− mice survived the infection under conditions in which 62% of the WT mice fully recovered (p < 0.05). These results indicated that GITR can protect mice from death in a lethal influenza model.

FIGURE 6.
FIGURE 6.

GITR on CD8 T cells augments viral clearance and protects against mortality during severe respiratory influenza infection. A, Mouse survival following influenza A/PR8 infection: WT and GITR−/− mice were infected with influenza A/PR8. Mice were monitored postinfection and sacrificed when moribund. Data are pooled from two independent experiments, each with six to seven mice per group. B–F, One thousand CD45.1 OT-I or CD45.1 GITR−/− OT-I cells were transferred into WT mice. One day later, mice were infected with X31-OVA (B, C) or PR8-OVA (D–F). Mice were monitored post–PR8-OVA infection and sacrificed when moribund. B and E, Body weight was monitored daily postinfection. Body weights were not read after day 7 for PR8-OVA–infected mice, as several were moribund and had to be sacrificed. Data in B are representative of three independent experiments, each using four to five mice per group. Data in E are pooled from two independent experiments, each having five to seven mice per group. C and F, Lung homogenates were analyzed for viral load at days 3 and 6 postinfection, and for X31-OVA on day 3, 6, and 8. At each time point, a minimum of five mice per group was used.

To further investigate whether GITR’s protective function was due to its intrinsic role on CD8 T cells, we transferred WT or GITR-deficient naive OT-I T cells into mice and then challenged them with X31-OVA or influenza A/PR8-OVA (PR8-OVA) (18). The X31-OVA induces a mild respiratory disease whereby mice lose ∼5–10% of their body weight, but clear the virus by day 8 and completely recover. Under these circumstances, the absence of GITR on the T cells had no effect on initial weight loss or viral clearance and 100% of mice survived (Fig. 6B, 6C). In contrast, infection with PR8-OVA causes up to 30% weight loss and can be lethal in mice. Previous results from our laboratory had shown that transfer of just 1000 naive OT-I T cells to mice prior to infection with PR8-OVA can protect WT mice from death, whereas transfer of high numbers of T cells abrogates the protective effect due to immune pathology (37). Using this optimized T cell dose, we observed that 100% of the mice that received 1000 GITR−/− OT-I cells or no transferred cells succumbed to the infection. In contrast, 40% of the mice that received WT OT-I cells recovered and survived (Fig. 6D). Thus, the absence of GITR on CD8 T cell recapitulates the mouse survival defect after influenza infection seen when GITR is absent on all cells. Although the absence of GITR on the CD8 T cells had no effect on the initial weight loss (Fig. 6E) induced in response to the early innate response to the virus, the mice that received GITR−/− OT-I cells had a 5.6-fold (0.75 log) higher viral load by day 6 (Fig. 6F) and were visibly sicker at day 7–8 postinfection as compared with those that had received 1000 WT OT-I cells. Thus, CD8 T cell-intrinsic GITR can protect mice from death during severe respiratory influenza infection, but is dispensable during mild respiratory influenza infection.

Discussion

GITR is emerging as an important molecule for regulating CD8 T cell responses. Moreover, the use of agonistic anti-GITR Abs to augment immune responses in vivo is of interest as a therapeutic tool. In administering GITR agonists in a therapeutic context, it will be important to understand GITR’s mechanisms of action and its cellular targets. Until now, the intrinsic role of GITR on CD8 T cells in vivo has been unclear. In this study, we make use of transgenic GITR−/− OT-I T cells to dissect the intrinsic role of GITR on CD8 T cells during influenza infection. We demonstrate that GITR is required on the CD8 T cells for maximal CD8 T cell expansion. Moreover, GITR on the CD8 T cells is also required for the enhancement of their responses following systemic administration of agonistic anti-GITR Ab. Whereas GITR did not enhance CD8 T cell proliferation, it played a key role in CD8 T cell survival. Under conditions of severe respiratory influenza infection, the presence of GITR on CD8 T cells led to augmented viral clearance and protection of the mice from death.

We first examined the NP366–374-specific CD8 T cell response when GITR−/− or WT C57BL/6 mice were infected with a mild strain of influenza. GITR−/− mice, however, showed defects only in certain organs at certain times. The lack of concordance between the GITR−/− OT-I adoptive transfer model, in which defects were seen in all organs examined, and the complete knockout, which showed only a subset of these defects, is most likely due to indirect effects of the lack of GITR on other cell types. Whereas GITR is present on naive T cells with increased expression upon infection, GITR can also be expressed on dendritic cells, macrophages, NK cells, NKT cells, and B cells upon infection. Furthermore, it is well established that GITR is highly expressed on CD4+CD25+ Tregs and aids in their costimulation. It has been noted that immune responses are highly dependent on the ratio of effector T cells to Tregs in the local environment. Therefore, because GITR works to costimulate both effector and Tregs, it is possible that the defects seen in one organ and not the others are due in part to differences in the ratios of effector T cells to Tregs in the various organs. In addition, it has been shown that GITR can negatively regulate dendritic cell (DC) function. DCs from GITR−/− mice have been shown to more effectively stimulate IL-2 and IFN-γ production from CD4 T cells than GITR+/+ DCs (38). It is difficult, therefore, to interpret the role of GITR on CD8 T cells in a model in which GITR may be interacting on multiple cell types to influence the CD8 T cell response indirectly.

Using the GITR−/− OT-I adoptive transfer model, we showed profound recovery defects of GITR−/− CD8 T cells in both the primary and recall response. Having ruled out rejection, kinetic, proliferative, and homing differences between WT and GITR−/− OT-I T cells, we showed that GITR is essential for CD8 T cell survival. Consistent with this, we demonstrated that GITR signaling induces NF-κB, which in turn leads to induction of the prosurvival molecule Bcl-xL. Riccardi and colleagues (11) previously showed that GITR is required for CD28-mediated upregulation of Bcl-xL expression. In this study, we have observed that Bcl-xL expression is enhanced directly downstream of GITR-mediated NF-κB signaling, as early as 3 h after GITR ligation.

It is well established that GITR can activate NF-κB signaling (3). Recently, it was shown that several TNFR family members, including GITR, are capable of activating both the canonical and noncanonical NF-κB pathways (39, 40). In the current study, we show that the canonical NF-κB pathway is rapidly activated downstream of GITR signaling, requiring both TRAF2 and TRAF5. Our data are consistent with previous studies showing that TRAF5 is essential for CD8 T cell survival (41) and for maximal NF-κB activation downstream of GITR in CD4 T cells (32). There has been controversy, however, as to the role of TRAF2 in activating NF-κB downstream of GITR signaling. Kwon et al. (31) report TRAF2 to be a positive regulator of GITR-dependent NF-κB signaling, in agreement with our data and consistent with studies showing that TRAF2 and TRAF5 play redundant roles downstream of TNF signaling (42). Esparza and Arch (30), however, have reported that GITR uses TRAF2 in a novel fashion as an inhibitor of NF-κB activation. Both Kwon et al. and Esparza and Arch overexpressed TRAF2 and performed a NF-κB luciferase assay 24 h after GITR stimulation. This assay does not distinguish between activation of the canonical and noncanonical NF-κB pathways. As TRAF2 has been shown to be positive for activation of the canonical NF-κB pathway, but negative for activation of the noncanonical NF-κB pathway (43), the NF-κB luciferase assay readout would be a balance between these two roles and may depend on the levels of TRAF2 expression. Our study specifically tested the role of TRAF2 and TRAF5 in the canonical pathway of NF-κB activation downstream of GITR in primary T cells and showed that they have additive and positive effects.

TRAF1 has been shown to bind to GITR in a yeast–2-hybrid assay (30) and in overexpression studies (31); however, its role in GITR signaling in primary T cells has not been examined. We observed that TRAF1−/− OT-I CD8 T cells could initiate NF-κB signaling in a manner similar to WT cells, indicating that TRAF1 is dispensable for GITR-induced NF-κB activation. However, it is possible that TRAF1 has a redundant role in NF-κB signaling, which in its absence can be compensated for by TRAF2 and TRAF5.

Several studies have shown that the agonistic anti-GITR Ab, DTA-1, enhances both CD8 and CD4 T cell responses to viruses and tumors in vivo (3). It is not known, however, whether this is a direct consequence of GITR ligation on the T cells or whether it occurs indirectly through effects on other cell types. Earlier studies attributed the DTA-1–induced effector T cell expansion to an indirect mechanism whereby the Ab was targeting Tregs and attenuating their function (1, 2). Indeed, it has been shown that DTA-1 can cause the proliferation of Tregs, although whether this enhances or abrogates their suppression remains controversial (3, 24). DTA-1 can also costimulate effector T cells (9, 10) and cause them to become more refractory to Treg suppression (7). A few groups have attempted to use depletion studies to rule out the role of DTA-1 on various cellular subsets, such as Tregs, thereby suggesting a direct role for DTA-1 in T cell costimulation (44, 45). To our knowledge, however, no study has been able to directly address the question of whether GITR is required on CD8 T cells for DTA-1–induced expansion. By using GITR−/− OT-I T cells, we show that the CD8 T cells lacking GITR failed to expand in response to DTA-1, with responses similar to those of WT OT-I cells treated with rat IgG. Therefore, we conclusively show that DTA-1 ligation of GITR on CD8 T cells is essential for maximal CD8 T cell responses to influenza, and that in this model DTA-1 ligation on other cell types, such as Tregs, does not play a major role.

In apparent contrast to the current study, a recent paper demonstrated that transgenic expression of GITRL on B cells expands CD4 T cells, but not CD8 T cells in mice (46). This might reflect the failure of CD8 T cells and B cells to efficiently interact in vivo.

We demonstrate that GITR is essential for the survival of influenza-specific CD8 T cells, with little effect on proliferation or effector function per cell. In contrast, other studies have shown that administration of agonistic GITR Abs in viral or cancer models can enhance both the effector function and proliferation of CD8 T cells (3). It is likely that the administration of the anti-GITR Ab gives a supraphysiological signal, which is much stronger than that of the endogenous GITRL in the OT-I model. Therefore, it is possible that the signal from the agonistic Ab would be sufficient to drive enhancement of proliferation and effector function, whereas the endogenous signal is not. It should be noted that in our studies, the addition of agonistic Abs to in vitro cultures with suboptimal peptide did not substantially enhance proliferation (data not shown) and therefore the effects of Ab may depend on the dosage and administration schedule used. Physiologically, however, our study shows that GITR’s main role on CD8 T cells in vivo is in promoting their survival.

A striking difference was observed in the requirement for GITR on CD8 T cells for disease outcome in mild versus severe influenza infection. Although expansion of CD8 T cells during infection with X31-OVA is impaired when the transferred T cells lack GITR, the mice still readily handle this infection and fully recover with complete viral clearance that is indistinguishable from mice receiving WT T cells. This is likely because in the milder influenza infection model, the combination of endogenous CD4, CD8, and Ab responses is able to readily control the virus (47). In contrast, in a more severe infection model with PR8-OVA, transfer of 1000 WT T cells protected 40% of the mice from death, whereas 100% of mice that received either no cells or 1000 GITR-deficient T cells succumbed to the infection. This decreased survival of mice that received GITR-deficient T cells as compared with WT T cells was associated with a 6-fold higher viral load at day 6 of the infection. As the only difference between the two groups is the presence of GITR on the transferred T cells, this argues that GITR on the CD8 T cells contributes to viral clearance and mouse survival. It is of interest to note that the mouse survival defect observed in the adoptive transfer model with GITR−/− OT-I cells and PR8-OVA infection recapitulates the defect seen when the complete knockout GITR−/− mice are infected with unmodified PR8. This suggests that whereas GITR on other immune cell types may aid in the protection of mice from lethal influenza, GITR on CD8 T cells most likely plays a major protective role.

Our laboratory has previously shown that the TNF family ligand 4-1BBL is also required for survival to severe influenza virus, but dispensable during mild flu infection (37). In that study, however, it was not determined whether this was a T cell-intrinsic effect. It has been suggested that the large number of TNF family receptors and ligands may have arisen by gene duplication, followed by diversification (48). The finding that GITR and 4-1BB costimulatory pathways are critical for mouse survival during severe influenza infection leads us to speculate that survival against severe viral infections may have contributed to the accumulation of TNF/TNFR costimulators during evolution. In contrast, there may also be circumstances in which such increased responses are pathological (49).

In summary, this study extensively dissects the role of GITR on CD8 T cells during viral infection. We show that GITR is essential for maximal primary and secondary CD8 T cell responses to influenza. GITR is required for the survival of CD8 T cells, and GITR signaling induces TRAF2- and TRAF5-mediated NF-κB activation, leading to enhanced Bcl-xL protein expression. Moreover, during severe influenza infection, GITR on CD8 T cells can play a critical role in augmenting viral clearance and decreasing mouse mortality. In addition, we demonstrate that GITR is required on CD8 T cells for agonistic anti-GITR Ab-mediated CD8 T cell expansion in response to influenza virus, thus providing a mechanism for the potential immunotherapeutic use of anti-GITR agonistic Ab.

Acknowledgments

We thank Peter Doherty and Paul Thomas for providing influenza A/HK-X31-OVA and influenza A/PR8-OVA; the National Institute of Allergy and Infectious Diseases tetramer facility for MHC I tetramers; Birinder Ghumman and Thanuja Ambagala for technical assistance; and Jennifer Gommerman for helpful comments.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • The online version of this article contains supplemental material.

  • This work was supported by Grant #84419 from the Canadian Institutes of Health Research (CIHR) to T.H.W. L.M.S. is funded by a fellowship from the Fonds de la Recherche en Santé du Québec. G.H.Y.L. is funded by a CIHR Doctoral Award. T.H.W. holds the Sanofi Pasteur Chair in Human Immunology at the University of Toronto.

  • Abbreviations used in this paper:

    DC
    dendritic cell
    GITR
    glucocorticoid-induced TNFR-related protein
    GITRL
    ligand for GITR
    HAU
    hemagglutinin units
    i.n.
    intranasal
    NP
    nucleoprotein
    PDTC
    1-pyrrolidinecarbodithioic acid
    siRNA
    small interfering RNA
    TCID50
    tissue culture infectious dose 50
    TRAF
    TNFR-associated factor
    Treg
    regulatory T cell
    WT
    wild-type.

  • Received June 9, 2010.
  • Accepted October 8, 2010.

References

  1. ↵
    1. McHugh R. S.,
    2. M. J. Whitters,
    3. C. A. Piccirillo,
    4. D. A. Young,
    5. E. M. Shevach,
    6. M. Collins,
    7. M. C. Byrne
    . 2002. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16: 311–323.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Shimizu J.,
    2. S. Yamazaki,
    3. T. Takahashi,
    4. Y. Ishida,
    5. S. Sakaguchi
    . 2002. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3: 135–142.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Nocentini G.,
    2. C. Riccardi
    . 2009. GITR: a modulator of immune response and inflammation. Adv. Exp. Med. Biol. 647: 156–173.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Kim W. J.,
    2. E. M. Bae,
    3. Y. J. Kang,
    4. H. U. Bae,
    5. S. H. Hong,
    6. J. Y. Lee,
    7. J. E. Park,
    8. B. S. Kwon,
    9. K. Suk,
    10. W. H. Lee
    . 2006. Glucocorticoid-induced tumour necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques. Immunology 119: 421–429.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Liu B.,
    2. Z. Li,
    3. S. P. Mahesh,
    4. S. Pantanelli,
    5. F. S. Hwang,
    6. W. O. Siu,
    7. R. B. Nussenblatt
    . 2008. Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis. J. Biol. Chem. 283: 8202–8210.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Baltz K. M.,
    2. M. Krusch,
    3. T. Baessler,
    4. B. J. Schmiedel,
    5. A. Bringmann,
    6. P. Brossart,
    7. H. R. Salih
    . 2008. Neutralization of tumor-derived soluble glucocorticoid-induced TNFR-related protein ligand increases NK cell anti-tumor reactivity. Blood 112: 3735–3743.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Stephens G. L.,
    2. R. S. McHugh,
    3. M. J. Whitters,
    4. D. A. Young,
    5. D. Luxenberg,
    6. B. M. Carreno,
    7. M. Collins,
    8. E. M. Shevach
    . 2004. Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J. Immunol. 173: 5008–5020.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Nishikawa H.,
    2. T. Kato,
    3. M. Hirayama,
    4. Y. Orito,
    5. E. Sato,
    6. N. Harada,
    7. S. Gnjatic,
    8. L. J. Old,
    9. H. Shiku
    . 2008. Regulatory T cell-resistant CD8+ T cells induced by glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res. 68: 5948–5954.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Ronchetti S.,
    2. O. Zollo,
    3. S. Bruscoli,
    4. M. Agostini,
    5. R. Bianchini,
    6. G. Nocentini,
    7. E. Ayroldi,
    8. C. Riccardi
    . 2004. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur. J. Immunol. 34: 613–622.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Tone M.,
    2. Y. Tone,
    3. E. Adams,
    4. S. F. Yates,
    5. M. R. Frewin,
    6. S. P. Cobbold,
    7. H. Waldmann
    . 2003. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc. Natl. Acad. Sci. USA 100: 15059–15064.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Ronchetti S.,
    2. G. Nocentini,
    3. R. Bianchini,
    4. L. T. Krausz,
    5. G. Migliorati,
    6. C. Riccardi
    . 2007. Glucocorticoid-induced TNFR-related protein lowers the threshold of CD28 costimulation in CD8+ T cells. J. Immunol. 179: 5916–5926.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. He H.,
    2. R. J. Messer,
    3. S. Sakaguchi,
    4. G. Yang,
    5. S. J. Robertson,
    6. K. J. Hasenkrug
    . 2004. Reduction of retrovirus-induced immunosuppression by in vivo modulation of T cells during acute infection. J. Virol. 78: 11641–11647.
    OpenUrlAbstract/FREE Full Text
    1. La S.,
    2. E. Kim,
    3. B. Kwon
    . 2005. In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1. Exp. Mol. Med. 37: 193–198.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Suvas S.,
    2. B. Kim,
    3. P. P. Sarangi,
    4. M. Tone,
    5. H. Waldmann,
    6. B. T. Rouse
    . 2005. In vivo kinetics of GITR and GITR ligand expression and their functional significance in regulating viral immunopathology. J. Virol. 79: 11935–11942.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Ronchetti S.,
    2. G. Nocentini,
    3. C. Riccardi,
    4. P. P. Pandolfi
    . 2002. Role of GITR in activation response of T lymphocytes. Blood 100: 350–352.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Tsitsikov E. N.,
    2. D. Laouini,
    3. I. F. Dunn,
    4. T. Y. Sannikova,
    5. L. Davidson,
    6. F. W. Alt,
    7. R. S. Geha
    . 2001. TRAF1 is a negative regulator of TNF signaling: enhanced TNF signaling in TRAF1-deficient mice. Immunity 15: 647–657.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Sabbagh L.,
    2. C. C. Srokowski,
    3. G. Pulle,
    4. L. M. Snell,
    5. B. J. Sedgmen,
    6. Y. Liu,
    7. E. N. Tsitsikov,
    8. T. H. Watts
    . 2006. A critical role for TNF receptor-associated factor 1 and Bim down-regulation in CD8 memory T cell survival. Proc. Natl. Acad. Sci. USA 103: 18703–18708.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Jenkins M. R.,
    2. R. Webby,
    3. P. C. Doherty,
    4. S. J. Turner
    . 2006. Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting. J. Immunol. 177: 2917–2925.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Pulle G.,
    2. M. Vidric,
    3. T. H. Watts
    . 2006. IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival. J. Immunol. 176: 2739–2748.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Cottey R.,
    2. C. A. Rowe,
    3. B. S. Bender
    . 2001. Influenza virus. Curr. Protoc. Immunol. Chapter 19:Unit 19.11.
  21. ↵
    1. Watts T. H.
    2005. TNF/TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol. 23: 23–68.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Croft M.
    2009. The role of TNF superfamily members in T-cell function and diseases. Nat. Rev. Immunol. 9: 271–285.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Croft M.,
    2. L. M. Bradley,
    3. S. L. Swain
    . 1994. Naive versus memory CD4 T cell response to antigen: memory cells are less dependent on accessory cell costimulation and can respond to many antigen-presenting cell types including resting B cells. J. Immunol. 152: 2675–2685.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Shevach E. M.,
    2. G. L. Stephens
    . 2006. The GITR-GITRL interaction: co-stimulation or contrasuppression of regulatory activity? Nat. Rev. Immunol. 6: 613–618.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Kanamaru F.,
    2. P. Youngnak,
    3. M. Hashiguchi,
    4. T. Nishioka,
    5. T. Takahashi,
    6. S. Sakaguchi,
    7. I. Ishikawa,
    8. M. Azuma
    . 2004. Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J. Immunol. 172: 7306–7314.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Savill J.,
    2. V. Fadok,
    3. P. Henson,
    4. C. Haslett
    . 1993. Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14: 131–136.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Esparza E. M.,
    2. R. H. Arch
    . 2005. Glucocorticoid-induced TNF receptor functions as a costimulatory receptor that promotes survival in early phases of T cell activation. J. Immunol. 174: 7869–7874.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Chen C.,
    2. L. C. Edelstein,
    3. C. Gélinas
    . 2000. The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L). Mol. Cell. Biol. 20: 2687–2695.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Arron J. R.,
    2. M. C. Walsh,
    3. Y. Choi
    . 2002. TRAF-mediated TNFR-family signaling. Curr. Protoc. Immunol. Chapter 11:Unit 11.19D.
  30. ↵
    1. Esparza E. M.,
    2. R. H. Arch
    . 2005. Glucocorticoid-induced TNF receptor, a costimulatory receptor on naive and activated T cells, uses TNF receptor-associated factor 2 in a novel fashion as an inhibitor of NF-kappa B activation. J. Immunol. 174: 7875–7882.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Kwon B.,
    2. K. Y. Yu,
    3. J. Ni,
    4. G. L. Yu,
    5. I. K. Jang,
    6. Y. J. Kim,
    7. L. Xing,
    8. D. Liu,
    9. S. X. Wang,
    10. B. S. Kwon
    . 1999. Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand. J. Biol. Chem. 274: 6056–6061.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Esparza E. M.,
    2. T. Lindsten,
    3. J. M. Stockhausen,
    4. R. H. Arch
    . 2006. Tumor necrosis factor receptor (TNFR)-associated factor 5 is a critical intermediate of costimulatory signaling pathways triggered by glucocorticoid-induced TNFR in T cells. J. Biol. Chem. 281: 8559–8564.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Arron J. R.,
    2. Y. Pewzner-Jung,
    3. M. C. Walsh,
    4. T. Kobayashi,
    5. Y. Choi
    . 2002. Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling. J. Exp. Med. 196: 923–934.
    OpenUrlAbstract/FREE Full Text
    1. Sabbagh L.,
    2. G. Pulle,
    3. Y. Liu,
    4. E. N. Tsitsikov,
    5. T. H. Watts
    . 2008. ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo. J. Immunol. 180: 8093–8101.
    OpenUrlAbstract/FREE Full Text
    1. Wicovsky A.,
    2. F. Henkler,
    3. S. Salzmann,
    4. P. Scheurich,
    5. C. Kneitz,
    6. H. Wajant
    . 2009. Tumor necrosis factor receptor-associated factor-1 enhances proinflammatory TNF receptor-2 signaling and modifies TNFR1-TNFR2 cooperation. Oncogene 28: 1769–1781.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Xie P.,
    2. B. S. Hostager,
    3. M. E. Munroe,
    4. C. R. Moore,
    5. G. A. Bishop
    . 2006. Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling. J. Immunol. 176: 5388–5400.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Lin G. H.,
    2. B. J. Sedgmen,
    3. T. J. Moraes,
    4. L. M. Snell,
    5. D. J. Topham,
    6. T. H. Watts
    . 2009. Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease. J. Immunol. 182: 934–947.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Vecchiarelli A.,
    2. E. Pericolini,
    3. E. Gabrielli,
    4. M. Agostini,
    5. F. Bistoni,
    6. G. Nocentini,
    7. E. Cenci,
    8. C. Riccardi
    . 2009. The GITRL-GITR system alters TLR-4 expression on DC during fungal infection. Cell. Immunol. 257: 13–22.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Bonizzi G.,
    2. M. Karin
    . 2004. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25: 280–288.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Hauer J.,
    2. S. Püschner,
    3. P. Ramakrishnan,
    4. U. Simon,
    5. M. Bongers,
    6. C. Federle,
    7. H. Engelmann
    . 2005. TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs. Proc. Natl. Acad. Sci. USA 102: 2874–2879.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Kraus Z. J.,
    2. J. S. Haring,
    3. G. A. Bishop
    . 2008. TNF receptor-associated factor 5 is required for optimal T cell expansion and survival in response to infection. J. Immunol. 181: 7800–7809.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Tada K.,
    2. T. Okazaki,
    3. S. Sakon,
    4. T. Kobarai,
    5. K. Kurosawa,
    6. S. Yamaoka,
    7. H. Hashimoto,
    8. T. W. Mak,
    9. H. Yagita,
    10. K. Okumura,
    11. et al
    . 2001. Critical roles of TRAF2 and TRAF5 in tumor necrosis factor-induced NF-kappa B activation and protection from cell death. J. Biol. Chem. 276: 36530–36534.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Vallabhapurapu S.,
    2. M. Karin
    . 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27: 693–733.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Cohen A. D.,
    2. A. Diab,
    3. M. A. Perales,
    4. J. D. Wolchok,
    5. G. Rizzuto,
    6. T. Merghoub,
    7. D. Huggins,
    8. C. Liu,
    9. M. J. Turk,
    10. N. P. Restifo,
    11. et al
    . 2006. Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity. Cancer Res. 66: 4904–4912.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Ramirez-Montagut T.,
    2. A. Chow,
    3. D. Hirschhorn-Cymerman,
    4. T. H. Terwey,
    5. A. A. Kochman,
    6. S. Lu,
    7. R. C. Miles,
    8. S. Sakaguchi,
    9. A. N. Houghton,
    10. M. R. van den Brink
    . 2006. Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity. J. Immunol. 176: 6434–6442.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. van Olffen R. W.,
    2. N. Koning,
    3. K. P. van Gisbergen,
    4. F. M. Wensveen,
    5. R. M. Hoek,
    6. L. Boon,
    7. J. Hamann,
    8. R. A. van Lier,
    9. M. A. Nolte
    . 2009. GITR triggering induces expansion of both effector and regulatory CD4+ T cells in vivo. J. Immunol. 182: 7490–7500.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Thomas P. G.,
    2. R. Keating,
    3. D. J. Hulse-Post,
    4. P. C. Doherty
    . 2006. Cell-mediated protection in influenza infection. Emerg. Infect. Dis. 12: 48–54.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Ware C. F.
    2003. The TNF superfamily. Cytokine Growth Factor Rev. 14: 181–184.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Humphreys I. R.,
    2. G. Walzl,
    3. L. Edwards,
    4. A. Rae,
    5. S. Hill,
    6. T. Hussell
    . 2003. A critical role for OX40 in T cell-mediated immunopathology during lung viral infection. J. Exp. Med. 198: 1237–1242.
    OpenUrlAbstract/FREE Full Text
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