In this study we examined the role of costimulation in modulating the activities of therapeutically administered memory T cells during the clearance of a persistent viral infection. Our results revealed that administration of adoptive immunotherapy induced marked systemic changes in host APC distribution and number. Within 3 days, DCs as well as monocytes/macrophages increased systemically in both lymphoid and nonlymphoid tissues. It was shown previously using CD11c-DTR carrier mice that successful adoptive immunotherapy requires host DCs (15
). We, therefore, examined the expression of costimulatory molecules on this potent APC population and observed that DCs in carrier mice upregulated CD80 and CD86 with kinetics that coincided with their numerical increase systemically. Blockade of these costimulatory molecules in vivo using CTLA-4-Fc resulted in a significantly reduced expansion of memory CD8+
T cells, decreased antiviral effector functions (i.e., cytokine-producing ability), and a delayed ability to clear an established persistent viral infection. Interestingly, our finding that costimulation blockade did not impede memory T-cell responses in vitro indicates that the in vivo environment imposes an additional set of constraints on antiviral memory T cells that cannot be adequately assessed through in vitro experimentation alone. It is important to note that the virus was cleared despite the impact on secondary expansion and function. These data indicate that an adoptive immunotherapeutic regimen consisting of antiviral T cells can be muted without necessarily impacting pathogen clearance. This fact was confirmed in late costimulation blockade experiments, which revealed that secondary memory T-cell expansion could be halved without impacting antiviral function or viral clearance kinetics. In concert, these data demonstrate that sustained costimulation is important in optimizing therapeutically administered memory T cells and that costimulation blockade can be used as a means to modulate or “apply the brakes” to an overly robust response without disrupting the ability to ultimately purge a persistent virus.
The landmark studies of Volkert and colleagues first revealed that memory immune cells could be used to eradicate an established persistent viral infection (29
), and it was demonstrated subsequently that the kinetics of this memory T-cell-dependent process differed between tissues (1
). LCMV carrier mice provide an excellent in vivo paradigm to uncover factors that modulate memory T-cell responses throughout the body. In this study we focused on the importance of costimulation using an inhibitor that is approved for clinical use, CTLA-4-Fc (16
). One of the original studies to suggest LCMV-specific memory T cells do not require costimulation was conducted in CD28-deficient mice (27
). CD28-deficient mice are able to clear LCMV Armstrong, and CD28-deficient mice that clear LCMV Armstrong are not susceptible to LCMV-induced meningitis or infection with immunosuppressive LCMV clone 13, suggesting normal memory T-cell formation. However, because the immune response to LCMV is so robust (17
), these assessments conducted in CD28-deficient mice would not have been expected to reveal a role for costimulation in antiviral memory T-cell function. In fact, it was subsequently revealed in different models of viral rechallenge that costimulation does in fact facilitate the activities of memory T cells (3
Here we set out to examine whether memory T cells used therapeutically do indeed require costimulatory molecules to purge an already persistent viral infection. We thought that the taxing and systemic antigenic environment found in LCMV carrier mice would provide a very stringent system to explore the absolute requirements of memory T cells because the experimental end point is total body viral eradication. One of the most interesting findings in our study is that costimulation blockade can be used as a means to fine tune an immunotherapeutic regimen consisting of memory T cells without preventing viral clearance. Both early and late costimulation blockade reduced the secondary expansion of memory T cells, but neither treatment completely prevented the therapeutic clearance of the persistent viral infection. Early (but not late) costimulation blockade also interfered with the ability of therapeutic memory T cells to produce antiviral cytokines but did not interfere with activation status or ability to produce IL-2. This knowledge is important for the treatment of persistent viral infections in humans, as it suggests that an overly robust and potentially injurious immunotherapeutic regimen can be dampened using a short treatment with a costimulation blocker. This is particularly important in light of a recent study demonstrating that costimulation blockade can reduce the immunopathology caused by memory CD4+
T cells in the lungs of mice rechallenged with influenza virus (28
). The upside of using CTLA-4-Fc treatment is that memory T-cell proliferation and function can be reduced without preventing the desired outcome of viral clearance. The fact that viral clearance is still attained suggests that the quantity of memory T cells that expand under normal conditions is well above the number required for clearance of a persistence viral infection. A fourfold reduction in the absolute number of antiviral CTL (as we observed when CTLA-4-Fc was administered 1 day before immunotherapy) is enough to delay but not prevent clearance, and a twofold reduction in CTL number (observed when CTLA-4-Fc was given on day 4) did not alter viral kinetics at all. These data, considered in the context of the aforementioned influenza study (28
), suggest that immunotherapeutic regimens can be finely tuned using CTLA-4-Fc to minimize the pathology induced by antiviral T cells while still preserving their ability to purge virus.
In our studies we also observed that costimulation blockade impeded memory T-cell proliferation in vivo but not in vitro. This difference is not surprising given the abundance and diversity of infected targets in LCMV carrier mice, an environment that simply cannot be replicated in a cell culture system. Previous in vitro studies by Croft et al. led to the conclusion that memory T cells are less dependent on costimulation than their naïve counterparts and respond to a broader range of APC types (4
). However, the consensus of several recent in vivo studies is that DCs represent an important bottleneck in primary and secondary responses (2
) and that costimulation does play an important role in the reactivation antiviral memory T cells (3
). Imposing this bottleneck makes sense evolutionarily, as memory CD8+
T cells receiving equivalent reactivation instructions from every MHC class I-bearing cell in the body could easily give rise to fatal immunopathology.
In conclusion, our findings not only demonstrate an important role for costimulation in optimizing the therapeutic clearance of a persistent viral infection, but they also offer new possibilities to those performing adoptive immunotherapy in a clinical setting. Costimulation blockade can be used before or following the therapeutic administration of antiviral T cells to modulate the robustness of the response. This intervention could be used to dull an overly aggressive therapeutic response and potentially limit undesirable immunopathology without preventing the eventual clearance of the persistent viral infection.