Cutting Edge: Ligation of the Glucocorticoid-Induced TNF Receptor Enhances Autoreactive CD4⁺ T Cell Activation and Experimental Autoimmune Encephalomyelitis

Materials and Methods

Results

We previously reported a functional role for CD4⁺CD25⁺ T_R cells in regulating the onset and progression of EAE such that T_R cell supplementation ameliorated clinical disease severity and CNS inflammation in both active and adoptive EAE (8). To further investigate the protective role of T_R cells during EAE, mice were treated with five 1-mg doses of anti-GITR mAb (DTA-1) to inactivate the T_R cell population (4, 5) at times spanning the induction and preacute disease phases. Anti-GITR-treated mice displayed a 0% survival rate upon reaching the “onset” phase of clinical disease (Fig. 1⇓A). This was an unexpected result, because the typical survival rate in this EAE system is 100%, suggesting that anti-GITR mAb treatment lethally enhanced the inflammatory response that normally precedes clinical disease onset. However, it was equally possible that the treatment regimen of repeated injections of a high dose of anti-GITR mAb produced a cumulative toxic effect. To test this possibility, similar studies were performed in which mice were immunized with a peptide known to lack inflammatory potential in SJL mice, OVA_323–339, in combination with anti-GITR mAb treatments. There was a 100% survival rate in OVA_323–339-primed mice receiving the identical anti-GITR mAb treatment regimen (Fig. 1⇓B) suggesting a role for the autoreactive inflammatory response in anti-GITR-treated mice primed with PLP_139–151.

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FIGURE 1.

Anti-GITR Ab treatment exacerbates EAE clinical disease progression and autoreactive T cell effector function. A and B, Effect of high-dose anti-GITR mAb treatment on EAE progression. Mice received five i.p. injections (arrows) of either anti-GITR or rat IgG control Ab (1 mg/injection) on alternating days either (A) beginning with the day of active EAE induction by s.c. injection of PLP_139–151/CFA or (B) the day of s.c. injection of OVA_323–339/CFA. Data are presented as the daily percent survival and are representative of three separate experiments. C–F, Effect of moderate-low dose anti-GITR mAb treatment on EAE progression. Beginning with the day of active EAE induction by s.c. injection of PLP_139–151/CFA, mice received four injections of anti-GITR mAb (100 or 500 μg/injection) on alternating days and were followed for clinical disease (C). D, Effect of anti-GITR mAb treatment on PLP_139–151-specific T cell proliferation during EAE. Data are presented as the mean cpm and are representative of three separate experiments. E and F, Effect of anti-GITR mAb on the frequency of PLP_139–151-specific Th1 (IFN-γ) and Th2 (IL-4) cells during EAE as measured by ELISPOT. Data are presented the number of PLP_139–151-specific IFN-γ (E) and IL-4 (F)-secreting cells per 10 × 10⁵ LN cells. Data presented are representative of three separate experiments. Significant differences from naive (∗) or control (∗∗), p < 0.01.

In light of the detrimental effect on survival following high dose anti-GITR treatment, similar experiments were performed with a lower dose of anti-GITR mAb. Mice treated with 500 μg of anti-GITR mAb displayed a significantly exacerbated clinical disease severity in the absence of any observed mortality (Fig. 1⇑C). In agreement with clinical disease severity, anti-GITR mAb treatment enhanced the level of PLP_139–151-specific T cell proliferation in both the LNs (Fig. 1⇑D) and spleen (data not shown). Anti-GITR mAb treatment also significantly increased the frequency of both PLP_139–151-specific Th1- (IFN-γ; Fig. 1⇑E) and Th2- (IL-4; Fig. 1⇑F) cytokine-producing cells, suggesting that anti-GITR mAb treatment functioned to generally enhance the autoantigen-specific immune response during EAE.

It is believed that the clinical symptoms of both EAE and multiple sclerosis manifest as a direct result of CNS inflammation and subsequent myelin damage apparently from the direct and indirect effects of chemokines (10) and proinflammatory cytokines such as IFN-γ and lymphotoxin/TNFβ (11, 12). We have previously shown that supplementation of the T_R cell population prevents CNS inflammation and ameliorates clinical disease severity potentially via effects on both autoreactive T cell activation and cell trafficking (8). Therefore, we next determined the effect of anti-GITR mAb treatment on the level of CNS inflammation at times corresponding to the peak of the acute clinical disease. In agreement with our previous findings, the level of CNS inflammation correlated with the degree of clinical disease severity such that anti-GITR mAb specifically increased the extent of CD4⁺ T cell infiltration within the CNS (Fig. 2⇓), without any discernable effects on either the CD8⁺ or B220⁺ cell populations which are usually not detected at high levels within the CNS of SJL mice with EAE. Taken together, these findings suggest that anti-GITR mAb treatment enhances autoreactive T cell activation and homing to the CNS resulting in an exacerbated level of clinical disease possibly via the functional inactivation of T_R cell populations.

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FIGURE 2.

Anti-GITR mAb treatment increases CNS inflammation during EAE. Spinal cord tissues from mice, receiving either saline, PLP_139–151, or PLP_139–151 + anti-GITR mAb (500 μg/injection on days 0, 2, 4, and 6 postimmunization), were examined on days 15–20 post-disease induction for the presence of CD4⁺, CD8⁺, and B220⁺ cells (red). Tissues were also counterstained with 4′,6′-diamidino-2-phenylindole (blue). No positive staining was observed in isotype-matched control samples (data not shown); ×100 magnification.

As previously discussed, anti-GITR mAb treatment is believed to specifically target and block the suppressive function of T_R cells (4, 5). Therefore, we reasoned that T_R cell depletion before EAE induction should result in enhanced clinical disease severity similar to that seen in mice treated with anti-GITR mAb. To our surprise, T_R cell depletion failed to influence the severity of acute clinical disease in our model system (Fig. 3⇓A). In addition, we found that SJL mice depleted of T_R cells and subsequently treated with anti-GTIR mAb surrounding PLP_139–151/CFA priming displayed enhanced clinical disease (Fig. 3⇓B) suggesting that anti-GITR has direct effects on cells other than CD4⁺CD25⁺ T_R cells. To further investigate the possibility that the anti-GITR mAb may target additional cell types, we examined the level of GITR expression on CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells. In agreement with previous findings (4), GITR was predominantly expressed on resting CD4⁺CD25⁺ T cells with only a small percentage of resting CD4⁺CD25⁻ T cells expressing detectable levels of the receptor (Fig. 4⇓A). However, GITR expression was rapidly up-regulated on the surface of CD4⁺CD25⁻ T cells within 24 h of activation by anti-CD3 mAb (Fig. 4⇓B) or following stimulation of naive PLP_139–151 TCR transgenic (Tg)⁺ T cells by Ag (data not shown). Interestingly, the observed up-regulation of GITR expression on CD4⁺ T cells correlated with their level of CD69 and CD25, both markers of T cell activation, and these experiments have also been repeated using T_R cell-depleted cultures (data not shown). To confirm the activation state dependency of GITR expression on CD4⁺CD25⁻ effector T cells, CD4⁺CD25⁻ T cells were also stimulated in the presence of F(ab′)₂ of the anti-CD3 Ab (CD3-F(ab′)₂) which blocks TCR-induced activation of CD4⁺ T cells.⁴ As expected, CD3-F(ab′)₂ blocked CD4⁺ T cell activation as determined by CD25, CD54, CD62L, and CD69 expression (data not shown), as well as activation-induced GITR expression (Fig. 4⇓C), indicating that GITR is a phenotypic marker of CD4⁺ T cell activation.

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FIGURE 3.

T_R cell depletion fails to influence EAE disease progression. A, SJL mice were depleted of T_R cells by anti-CD25 mAb injection (500 μg/injection i.p.) on days −4 and −2 before EAE induction by s.c. injection of PLP_139–151/CFA. Data are presented as the mean clinical disease score and are representative of three separate experiments. B, SJL mice were depleted of T_R cells by anti-CD25 mAb injection (500 μg/injection i.p.) on days −4 and −2 before EAE induction by s.c. injection of PLP_139–151/CFA. Mice received four injections of anti-GITR mAb (500 μg/injection) on alternating days and were followed for clinical disease.

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FIGURE 4.

Expression and functional role for GITR in CD4⁺CD25⁻ effector T cells. A–C, GITR Expression on CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells. A–C, LN cells isolated from naive mice were stained with Abs specific for CD4, CD25, CD62L, and GITR. Some cells were either activated with (B) anti-CD3 Ab (1 μg/10⁶ cells) or (C) anti-CD3 Ab + CD3-F(ab′)₂ (1 μg/10⁶ cells) for 24 h before immunofluorescence staining. Data are presented as the relative fluorescence intensity of GITR cells. All data are representative of three separate experiments. D–F, Effect of anti-GITR mAb ligation on CD4⁺CD25⁻ T cell proliferation. D, Naive SJL LN cells were depleted of CD4⁺CD25⁺ T_R cells before activation with anti-CD3 mAb (0.1 μg/10⁶ cells) in the presence of an increasing concentration of anti-GITR mAb. E, Naive SJL LN cells were depleted of CD4⁺CD25⁺ T_R cells before activation with increasing concentrations of anti-CD3 mAb in the presence of anti-GITR mAb (1 μg/10⁶ cells). F, LN cells isolated from naive 5B6 PLP_139–151 TCR transgenic mice were depleted of CD4⁺CD25⁺ T_R cells and activated with an increasing concentration of PLP_139–151 in the presence of an increasing concentration of anti-GITR mAb. All data are presented as the mean cpm and are representative of three separate experiments.

Because both activated CD4⁺ T cells and T_R cells expressed GITR, the possibility existed that anti-GITR mAb treatment may also directly influence CD4⁺ effector T cell function. To test this, we examined the effects of Ab-mediated cross-linking of GITR on CD4⁺ T cell proliferation in the absence of T_R cells. Activation of T_R cell-depleted LN (Fig. 4⇑D) or spleen (data not shown) cells in the presence of anti-GITR mAb increased the level of anti-CD3-induced T cell proliferation in a concentration-dependent manner. Interestingly, anti-GITR mAb treatment produced similar findings when added to T_R cell-depleted cultures of naive OVA_323–339-specific Tg T cells (DO11.10) activated with suboptimal (0.1 μg/ml), but not optimal, levels of Ag (1 μg/ml) (Fig. 4⇑E). Of direct relevance to the observed effects in our EAE model system, anti-GITR mAb treatment also enhanced the proliferation of T_R cell-depleted naive PLP_139–151-specific TCR Tg CD4⁺ T cells in a concentration-dependent manner (Fig. 4⇑F) suggesting that anti-GITR mAb treatment can enhance the effector function of both naive and previously activated CD4⁺ T cells (Fig. 1⇑D) in the absence of CD4⁺CD25⁺ T_R cells. Lastly, anti-GITR mAb treatment of T_R cell-depleted OVA_323–339-specific T cells also enhanced the level of cellular activation as determined by CD54, CD62L, and CD69 expression in a concentration-dependent manner (Fig. 5⇓), but failed to influence CD25 expression.

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FIGURE 5.

Anti-GITR mAb treatment enhances CD4⁺CD25⁻ T cell activation. LN cells were isolated from naive DO11.10 TCR transgenic mice and depleted of CD4⁺CD25⁺ T_R cells before activation in the presence of OVA_323–339 (10 μM) and an increasing concentration of anti-GITR mAb for 24 h before determination of the level of CD54, CD62L, CD69, and CD25 surface expression by flow cytometry. Data are presented as the relative fluorescence intensity of each activation marker on CD4⁺ T cells; MFI, mean fluorescence intensity.

Discussion

Consistent with previous reports showing that GITR is predominantly expressed on CD4⁺CD25⁺ T_R cells and that anti-GITR inactivates T_R cell functionality (4, 5), we found that treatment of SJL mice with anti-GITR mAb significantly exacerbated PLP_139–151-induced clinical EAE, CNS inflammation, and peptide-specific T cell responses (Figs. 1⇑ and 2⇑). However, our present findings also demonstrate that GITR is rapidly up-regulated on naive CD4⁺CD25⁻ T cells following either mitogen- or Ag-induced T cell activation and that anti-GITR enhanced the level of CD4⁺ T cell activation, proliferation, and cytokine production in the absence of T_R cells in vitro (Figs. 4⇑ and 5⇑). The expression pattern of GITR is thus similar to that of CD25 which is also preferentially expressed at higher levels on resting T_R cells, but quickly up-regulated on naive CD4⁺ T cells upon TCR activation, raising the possibility that CD25 and GITR expression may be regulated by similar mechanisms. Thus, it may be more appropriate to consider GITR expression as a marker of T cell activation rather than defining the T_R population.

We previously showed that T_R cell supplementation blocked the activation of both naive and previously activated autoreactive CD4⁺ T cells and protected against the initiation and progression of EAE (8). However, the question remains as to why anti-GITR-mediated T_R inactivation (Fig. 1⇑), but not anti-CD25-mediated T_R cell depletion, led to enhanced acute clinical EAE (Fig. 3⇑A). One possible explanation may be that the anti-CD25 depletion protocol used in the current studies simply down-regulated the level of CD25 expression on T_R cells rather than deplete this population. However, in vitro studies using the same depleting and detecting Ab clones did not result in significant down-regulation of CD25 expression on CD4⁺ T cells (data not shown). Regardless, the possibility exists that distinct mechanisms may function in vivo and current experiments are closely examining the fate of CD4⁺CD25⁺ T_R cells following anti-CD25 mAb injection. An alternative explanation for the failure of T_R cell depletion to influence the clinical disease progression in EAE may be the existence of redundant compensatory regulatory mechanisms in vivo including other regulatory cell populations. However, further phenotypic and functional characterization of these alternative regulatory populations is necessary to determine their contribution to immune homeostasis during EAE.

Of particular interest is our finding that ligation of GITR by anti-GITR mAb enhanced CD4⁺ T cell activation, proliferation, and cytokine production in the absence of T_R cells both in vivo (Fig. 3⇑B) and in vitro (Fig. 4⇑). Moreover, ligation of GITR appears to optimally augment CD4⁺ T cell activation in conditions of suboptimal TCR ligation. Therefore, in addition to previously reported effects of anti-GITR on inactivating T_R cell activity (4, 5), GITR ligation appears to directly enhance CD4⁺ T cell effector function by delivering a costimulatory signal thus lowering the signaling threshold of the TCR complex. Collectively, our findings suggest a dual functional role for GITR wherein GITR ligation both inactivates T_R cells and, at the same time, increases the activation and effector functions of CD4⁺CD25⁻ T cells, thus resulting in enhanced T cell-mediated immunity.