The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain-containing adapter inducing IFN-β are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1β are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC^-/- animals. Importantly, the early proliferation of Ag-specific CD4⁺ T cells was completely ablated after immunization in ASC^-/- animals. Moreover, numbers of Ag-specific CD4⁺ T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC^-/-, NLRP3^-/-, and IL-1R^-/- mice compared with wild-type mice and were completely ablated in TLR4^-/- animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4⁺ T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.

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