Persistent viruses, such as human immunodeficiency virus, cause major health problems worldwide and are extraordinarily difficult to clear following the establishment of persistence. Given the challenges associated with clearing persistent infections, it is important to develop and mechanistically understand therapeutic strategies that successfully achieve viral eradication without inducing permanent damage to the host. Studies using the lymphocytic choriomeningitis virus (LCMV) model system have demonstrated convincingly that a systemic persistent viral infection can be completely purged from a murine host by using a therapeutic approach referred to as adoptive immunotherapy. Remarkably, total body control of multiple persistent viral infections in both the mouse and humans can be achieved using adoptive immunotherapy. When mice are persistently infected at birth or in utero with LCMV (referred to as carrier mice), the virus establishes systemic persistence. Adult LCMV carrier mice are tolerant to the virus at the T cell level and thus are unable to eradicate the pathogen, which provides an excellent model to study immunotherapeutic regimens. Immunocytotherapy relies on the adoptive transfer of virus-specific memory CD8 and CD4 T cells from LCMV-immune donor mice into recipient carrier mice. Following the therapeutic administration of memory cells, LCMV is purged from most peripheral tissues of carrier mice in 14 days, whereas more than 100 days are required to clear virus from the central nervous system (CNS) and kidneys. Furthermore, successful viral clearance can be achieved with antiviral memory but not effector T cells. Thus, in addition to its proven therapeutic relevance, this model also provides a paradigm to understand factors that regulate memory T cells following secondary exposure to pathogens in vivo. Our laboratory is interested in mechanistically understanding how the immune system can be harnessed to resolve persistent viral infections, particularly those that take residence in the CNS. LCMV carrier mice provide an ideal model to study because every tissue, including the CNS, contains an abundance of viral antigen. Thus, following administration of therapeutic memory T cells, the mechanisms underlying clearance of a persistent viral infection can be studied in all tissues simultaneously. Clearance of the persistently infected CNS is of particular interest in LCMV carrier mice because neurons are the primary cell population infected within the brain parenchyma, and these cells are thought to express minimal to no MHC I. Nevertheless, adoptively transferred memory T cells achieve clearance of these cells with very little evidence of cellular injury. Our recent studies demonstrated that the success of adoptive immunotherapy in LCMV carrier mice relies on assistance from the recipients immune system. For example, we observed that migration of anti-viral cytotoxic lymphocytes (CTL) into the CNS coincided temporally with the arrival of recipient antigen-presenting cells (e.g. dendritic cells). We became particularly interested in dendritic cells (DCs) because they are not normally found in the uninflamed brain parenchyma; however, following adoptive immunotherapy, they not only migrated into the brain, but also presented viral peptides to therapeutic CTL. Using depletion studies, we revealed that recipient DCs were in fact required for the success of adoptive immunotherapy both in the periphery as well as the CNS. More recently, we exploited the involvement of DCs in this process by using a costimulation blocker approved for clinical use (i.e., CTLA-4-Fc) to modulate the activities of therapeutic memory T cells. We observed that within days of adoptive immunotherapy memory T cells induced the recruitment of DCs into the CNS as well as peripheral tissues. As expected, this migration was associated with an upregulation of the costimulatory molecules, CD80 and CD86, on the DCs. Previously, it was thought that memory T cells, unlike their nave counterparts, did not require costimulation to be reactivated. However, more recent studies, including ours, called this dogma into question by noting that memory T cells do in fact require costimulation for optimal proliferation and effector functions. Importantly, we demonstrated that costimulation blockade with CTLA-4-Fc could be used to control the proliferation of therapeutic memory T cells without impeding their ability to ultimately purge persistent virus. This finding has the potential to translate into an important therapeutic tool because memory T cells can cause severe (sometimes fatal) immunopathology. Lessening T cell numbers without impacting viral clearance should reduce unwanted immunopathology. We are presently in the process of following up on these exciting observations. The overall aim of this project is to define how therapeutic memory T cells coordinate with host innate immune cells to achieve clearance of the persistently infected CNS, particularly neurons. We demonstrated this past year that the host innate immune system is chronically stimulated by type I interferon production during a LCMV carrier state, and previous studies have demonstrated that type I interferons are required for the success of adoptive immunotherapy. We, therefore, propose that the dialogue between the innate and adaptive immune systems is critical for the eradication of a persistent viral infection. Using real time imaging approaches, we are now attempting to reveal the precise mechanism(s) by which the innate and adaptive immune systems purge neurons of virus. Despite the low level of MHC I expression, it is conceivable that CTL directly engage neurons. Alternatively, it is possible that CTL indirectly achieve clearance of neurons through their interactions with DCs. These hypotheses can be easily addressed by simultaneously visualizing in real time infected neurons, DCs, and CTL each labeled with distinct fluorescent tags. This will be accomplished by imaging the living brains of immunotherapy recipients using two photon microscopy. We are also interested in defining the mechanism(s) used by neurons to uniquely regulate the immune system and prevent unwanted immunopathology. We postulate that CTL can directly interact with CNS neurons but are uniquely regulated upon doing so. Harnessing regulatory mechanisms therapeutically might improve the outcome of CNS infections that have severe consequences in humans.