Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10–20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)γ production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNγ, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

IL-12 and TNF-α production by dendritic cells (DCs) is a critical step in the initiation of local inflammation and adaptive immune responses. We show in this study that a small molecule immune response modifier that is a Toll-like receptor 7 (TLR7) agonist induces IL-12 and TNF-α production from murine CD11c+CD11b+CD8− DCs, a subset not previously known for this activity. Stimulation of these DCs through TLR7 in vivo induces significant cytokine production even 12 h after initial stimulation, as well as migration of the DC into T cell zones of the lymphoid tissue. In contrast, stimulation through TLR4 and TLR9 induced IL-12 production predominantly from CD8+ DCs, consistent with previously published data. All TLR stimuli induced the increase in surface expression of the activation markers B7-1, B7-2, and class II in both CD8+ and CD8− DCs, demonstrating that CD8+ DCs do respond to TLR7-mediated stimuli. To date this is the only known stimuli to induce preferential cytokine production from CD8− DCs. Given the efficacy of TLR7 agonists as antiviral agents, the data collectively indicate that stimulation of CD8− DCs through TLR7 most likely plays a role in the generation of antiviral immune responses.