Invariant Natural Killer T (iNKT) cells are a unique subpopulation of lymphocytes that expresses an invariant T cell antigen receptor (TCR) alpha chain that is highly conserved between mice and humans. This unique TCR, and the conservation of its antigenic specificity, suggests that experiments performed in mouse models may have a particular relevance for human studies. These cells have been implicated in the responses to several bacteria that have glycolipid antigens that can engage their canonical TCR. Additionally, recent studies have demonstrated that iNKT cells have a role in the innate response to diverse microbes, including viruses, that do not encode antigens for their invariant TCR. Our preliminary studies have demonstrated that iNKT cells are activated in vitro to produce cytokines after culture with dendritic cell (DC) subtypes that have been infected with mouse cytomegalovirus (MCMV). Additionally, we have begun to identify the role of iNKT cells in vivo in response to infection with MCMV during the initiation of the innate immune response within 36 hours after infection. MCMV is a well characterized and studied virus and is ideal model pathogen for investigating how iNKT cells are activated following virus infection. The first aim in this proposal is to identify the in vivo roles of TLR9, IL-12, and CD1d-rnediated antigen presentation for the activation of iNKT cells by MCMV infection. Our extensive preliminary in vitro data indicates that DC capable of iNKT cells activation upon MCMV infections arise in Flt3 ligand (FltSL) derived DC cultures, but not in DC derived in GM-CSF. Activation in vitro requires TLR9 expression and the synthesis of IL-12, but in most experiments, did not require CD1d expression. These data suggest that recognition of self-antigens presented by CD1d is not absolutely necessary to activate iNKT cells in culture for 48 hrs. We will carry out experiments in vivo to identify the relevance of the data generated in vitro following MCMV infection. The second aim will identify the relevant DC type(s) that activate iNKT cells after MCMV infection. Since the FltSL derived DC culture is a heterogeneous mix of DC subtypes, we will purify DC subsets and identify those responsible for iNKT cell activation. This will enable us to define the mechanisms responsible for iNKT cell activation in vitro and in vivo. Our preliminary data suggests that plasmacytoid DC present in the FltSL DC cultures may contribute iNKT cell activation. Therefore, we will deplete these cells in vivo using a PDCA-1- specific antibody and assess whether this alters activation of iNKT cells upon MCMV infection in vivo. Additionally, we will test the ability of ex vivo purified DC subsets sequentially infected with MCMV to activate iNKT cells in vitro.