We report that giving an agonist anti-GITR antibody during immunization enhances both effector and memory CD8+
T-cell responses against defined self-antigens expressed by tumors and that these responses translate into improved rejection of a poorly immunogenic tumor. Although GITR ligation can abrogate Treg-mediated suppression and increase the proliferation of both CD4+
T cells in vitro
), less is known about the effects of GITR ligation in vivo
on T-cell function, particularly CD8+
T cells. Muriglan et al. (19
) showed that the proliferation of alloreactive CD8+ T cells following adoptive transfer into MHC-mismatched recipients was increased with in vivo
administration of anti-GITR antibody along with the severity of CD8+
T-cell–mediated GVHD. Giving DTA-1 in combination with adoptive transfer of viral antigen-specific CD8+
T cells into virus-infected mice led to greater production of IFN-γ and TNF and greater reduction of viral load than adoptive transfer alone (39
). Together with our results, these studies show that GITR ligation can lead to enhanced CD8+
T-cell effector function in vivo
whether the T-cell specificity is for an alloantigen, viral antigen, or self-antigen expressed by tumors.
We observed enhanced immunity when DTA-1 was given just before the second vaccination (day 6) but no effect or worsened tumor protection () and CD8+
T-cell responses (data not shown) when the antibody was given before the initial priming vaccination (day 1). GITR is expressed constitutively at high levels by CD4+
Tregs and at low levels on resting CD4+
T cells. Up-regulation of GITR begins 6 hours after TCR engagement in vitro
and peaks at 72 hours (13
). Considering the time required for antigen production, processing, and cross-presentation by APCs following DNA vaccination, it is likely that GITR expression on activated effector T cells is still high at the time of anti-GITR antibody administration on day 6. In contrast, ligation of GITR before the initial priming immunization, in the absence of any TCR stimulation, may not only fail to costimulate T cells during subsequent activation but also impair activation and expansion, perhaps by interfering with normal GITR-GITR ligand interactions between T cells and APCs important for priming T-cell responses (16
). Support for this hypothesis comes from Shimizu et al. (17
) who noted that the ability of DTA-1 to abrogate Treg suppression in coculture assays was only seen when DTA-1 was added to the culture together with anti-CD3 antibody and irradiated APCs. When effector cells were preincubated with DTA-1 followed by anti-CD3-mediated activation, Tregs still suppressed proliferation.
An alternative explanation is that early GITR ligation followed by repetitive restimulation with antigen may increase susceptibility of T cells to AICD. The effects of GITR ligation on apoptotic signals remain unclear, with reports of both antiapoptotic (via nuclear factor-κB activation; refs. 10
) and proapoptotic effects (via interactions with Siva; ref. 43
). What is clear is that the in vitro
effect of GITR ligation on CD4+
T-cell proliferation is most pronounced under conditions of suboptimal TCR stimulation regardless of the presence of Tregs. When strong TCR stimulation is combined with higher concentrations of anti-GITR antibody, T-cell proliferation can actually decrease (13
). This effect has also been seen in vivo
. Muriglan et al. (19
) showed that GITR ligation paradoxically ameliorated GVHD mediated by CD4+
T cells. DTA-1 treatment was inducing apoptosis in alloactivated CD4+
cells. Similarly, Valzasina et al. (21
) found that GVHD mediated by alloreactive CD4+CD25 T cells was exacerbated by a low DTA-1 dose (300 μg) but mitigated by a high dose (1,200 μg), again suggesting AICD of the alloreactive cell population with stronger GITR signaling. Our data show that DTA-1 given before the first, but not the second, immunization, increases the susceptibility of antigen-specific CD8+
T cells to AICD (). We are currently investigating whether this phenomenon is responsible for the differences observed with the administration of DTA-1 before the first immunization versus the second immunization.
The circumstances surrounding the initial priming of effector T cells profoundly influence the subsequent memory T-cell population. Factors important during priming include CD4+
T-cell help, the duration and strength of antigenic stimulation, costimulatory molecules, the magnitude of the initial response, and the expression of cytokines and cytokine receptors (e.g., interleukin-7 receptor α; refs. 36
). In addition, depleting Tregs either during the primary response or at the time of a recall response can enhance the magnitude and function of memory CD8+
T cells recognizing pathogens (38
), implying a role for Tregs in memory T-cell generation.
Our work shows that a single dose of agonist anti-GITR mAb during a primary immunization series increases not only the magnitude of the initial effector CD8+
T-cell response but also the persistence of functional antigen-specific T cells (). Moreover, GITR ligation during primary hgp100 DNA immunization led to a greater recall response following booster immunization 4 weeks later, with a higher proportion of gp100-specific CD8+
T cells expressing an activated effector/memory (CD44hi
) phenotype (). Ultimately, however, both recall CD8+
T-cell responses and tumor rejection after secondary boosting were weaker than at the peak of the effector response (4−5 days after the primary immunization series). This pattern of weak memory response is different from the typically robust recall responses against viral and bacterial pathogens (37
) and likely reflects inherent difficulties in maintaining a functional, nontolerized memory population specific for constitutively expressed self-antigens. Recall CD8+
T-cell responses were improved, however, by ligating GITR during both primary and booster vaccination, approaching levels induced during the peak effector response (). This combined approach also led to improved memory CD8+
T-cell responses against B16 melanoma ex vivo
() as well as significantly improved protection following late (day 33) tumor challenge after a boost (), showing that these difficulties can be partially overcome through this approach.
The question of which cell subpopulations the anti-GITR antibody is primarily acting upon is debated. Although the ability of GITR ligation to directly costimulate CD4+
T cells has been well documented, the notion that anti-GITR therapy renders CD4+
Tregs unable to suppress (17
) has recently been challenged by in vitro
data suggesting that GITR ligation allows effector T cells to resist suppression rather than directly affecting Tregs themselves (16
). In contradistinction, a recent study using DTA-1 in a GVHD model supports a direct effect of DTA-1 on Tregs (21
To address this issue, we depleted CD4+ cells during hgp100 DNA immunization and found that CD8+ T-cell responses were still enhanced by DTA-1 (), supporting a direct effect on CD8+ effector T cells. The magnitude of enhancement was lower than in non-CD4-depleted mice, implying that GITR signaling in one or more CD4+ populations may also play a role. The enhancement of CD8+ T-cell responses () and tumor immunity () by DTA-1 despite previous depletion of CD25+ cells also indicates that GITR ligation on naturally arising Tregs is not required for its effects. In addition, although we did not observe an additive effect of combined PC61 (anti-CD25 mAb) and DTA-1 treatment, this may be due to the concomitant depletion by PC61 of some effector CD4+ and CD8+ T cells that up-regulated CD25 following activation, diluting the direct costimulatory effect of DTA-1. We speculate that both blockade of Treg suppressive activity and costimulation of effector T cells are contributing to the enhancement of active immunization by DTA-1. To identify the cellular targets of DTA-1 in this system, it will be necessary to test the DTA-1 plus immunization strategy in mice reconstituted with various combinations of GITR-positive and GITR-negative T cells. We are currently backcrossing GITR−/− mice onto the C57BL/6 background for this purpose.
In conclusion, administration of an agonist anti-GITR mAb during DNA immunization against self-antigens expressed by tumors augments vaccine-induced CD8+
T-cell responses and immunity against an aggressive tumor and represents a potentially novel immunotherapeutic strategy against cancer. This approach can increase autoimmunity as well (; refs. 12
), requiring thoughtful choice of vaccination targets and careful monitoring for autoimmune toxicity. Finally, DTA-1 antibody given alone (without any vaccination) after tumor challenge can impede tumor growth and modestly increase tumor-induced T-cell responses (45
Thus, GITR ligation may provide a platform on which to build a successful combination immunotherapy designed to eradicate established tumors.