Ovalbumin was purchased from Sigma Corporation and contaminating LPS removed using a TritonX-114 LPS-detoxification methodology as previously described (52). memory against bacterial challenge withListeria monocytogenes. Together these data show the importance of assessing CD70 expression on DCs as a marker for the capacity of a given vaccine formulation to potently activate cellular immunity. Our data show that optimal induction of CD70 expression requires a coordinated activation of both innate (TLR, IFN, GalCer) and adaptive (CD40) signaling pathways. == Introduction == Toll-like receptor (TLR) activation elicits a pro-inflammatory response involving the induction of the transcription factor NFB and MAP kinases, leading to the production of pro-inflammatory cytokines such as TNF, IL-12, IL-6, and IL-1 by dendritic cells (DCs) and macrophages(1,2). Triggering of TLRs 3, 4, 7, and 9 also prospects to production of Type I Interferons (IFN), cytokines which have been shown to be important in the generation of adaptive immunity (310). Given the ability of TLRs to induce the production of these proinflammatory responses, TLR agonists are generally thought to be useful vaccine adjuvants (11). Indeed, TLR IWP-3 agonists are relatively successful at augmenting humoral immune responses (12), However, when used to enhance the generation of cellular immune responses, TLR agonists by themselves have largely been a disappointment (1322). Indeed, when used alone as a vaccine adjuvant, we previously showed that they are unable to generate cellular immune responses capable of protecting against viruses or other intracellular infections (23). The paucity with which TLR agonists initiate cellular immunity reveals the fact that the precise quantity and/or quality of DC maturation that promotes the highest magnitude T cell response is still TIAM1 poorly understood. In general there is a prevailing view that more is better. Beyond that however, a precise characterization of how much of which costimulatory molecules are required for optimal T cell growth is as of yet unknown, a IWP-3 somewhat surprising fact given the degree of effort that is put into investigating both DC and T cell activation. CD70 is usually a costimulatory marker expressed primarily by antigen presenting cells (APCs)(24). It is a member of the TNF ligand superfamily and binds to its receptor CD27, which is usually expressed mainly on T cells but IWP-3 is also expressed on B cells and NK cells. CD70 has been shown to be important for the priming of CD8 T cell responses(23,2532) and blockade of CD70/CD27 interactions often dramatically reduces the generated CD8 T cell response(23,25,26,2830). Importantly, CD70 has also been shown to be upregulated on APCs during some natural infections, and is known in this setting to be important for CD8 T cell priming(30). Additionally, the constitutive expression of CD70 on immature DCs is enough to overcome tolerance and primary CD8+ T cells that can infiltrate into tumor sites and induce tumor regression(27). Given the potency of CD70 for inducing cellular immunity, an established method for inducing its expression on DCs would be of great benefit for the purposes of vaccine development. Toward this end, we previously exhibited that immunization with both a TLR agonist and an agonistic antiCD40 antibody induces a degree of CD8+ T cell growth that is 1050 fold greater than that observed in response to immunization in the presence of either agonist alone(14,23). The magnitude of these T cell responses often matches or even exceeds that seen in response to infectious brokers such as LCMV orListeria monocytogenes(1821). When comparing the phenotype of dendritic cells stimulated with a TLR agonist, anti-CD40, or both, we observed that upregulation of the TNF ligand superfamily member CD70 on both CD8 and CD11b DC subsets was unique to only the combined TLR agonist/antiCD40 stimulus(23). The CD8 T cell response generated by the combined TLR agonist/antiCD40 stimulus was dependent on this DC CD70 expression, since blocking CD70 from its receptor CD27, dramatically reduced the CD8 T cell response. Thus, CD70 expressionin vivois uniquely regulated by the combined activation of a TLR and CD40. Here we show that this innate signaling.