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Flagellin is a highly effective adjuvant, but the cellular mechanism underlying this activity remains uncertain. More specifically, no consensus exists as to whether flagellin activates dendritic cells (DC) directly or indirectly. Intramuscular immunization with flagellin-OVA fusion protein resulted in enhanced in vivo T cell clustering in draining lymph nodes and IL-2 production by OVA-specific CD4(+) T cells. Immunization with flagellin-OVA also triggered greater levels of Ag-specific CD4(+) T cell proliferation than immunization with flagellin and OVA as separate proteins. To determine whether flagellin, in the context of a fusion protein with OVA, was acting directly on DC, we used a combination of CD4(+) T cell adoptive transfers and bone marrow chimera mice in which the presence or absence of potential tlr5(+/+) CD11c(+) cells was controlled by injection of diphtheria toxin. The Ag-specific CD4(+) T cell response in mice with CD11c(+) cells from a tlr5(-/-) background and mixed populations of all other hematopoietic cells was dramatically reduced in comparison to mice that had DC from tlr5(-/-) and wild-type backgrounds. Immunization of MyD88(-/-)tlr5(+/+) mice revealed that the enhanced response following immunization with flagellin-OVA is dependent on signaling via the TLR5-MyD88 pathway as well as enhanced Ag uptake and processing resulting from Ag targeting via TLR5. In summary, our data are consistent with the conclusion that direct stimulation of tlr5(+/+) CD11c(+) cells is necessary for the adjuvant activity of a flagellin fusion protein and that this adjuvant effect requires signaling through TLR5.

Direct Stimulation of tlr5+/+ CD11c+ Cells Is Necessary for the Adjuvant Activity of Flagellin