The goal of this proposal is to uncover the in vivo mechanisms by which STING (Stimulator of interferon genes) mediates the mucosal vaccine adjuvant activity of cyclic di-GMP (CDG). Protective mucosal immune responses are most effectively induced by mucosal immunization. However, most of the currently approved human vaccines are administered systemically and generally fail to elicit effective mucosal immunity. Live attenuated mucosal vaccines present safety and acceptability issues while purified antigens are generally poor immunogenic when administered by the mucosal route. CDG exhibits potent mucosal immunogenicity thus, has been explored as a promising mucosal vaccine adjuvant. The mechanism by which CDG executes its mucosal adjuvant activity is unknown, which hinders the further development of CDG as an efficacious mucosal adjuvant. STING, also known as MPYS/MITA, is a receptor for CDG. We recently showed that STING-/- mice fail to generate Ag-specific antibody production or TH1-TH2-TH17 T cell response after intranasal CDG/Ag immunization. We further discovered that STING-dependent TNF-a is essential for CDG adjuvant activity in vivo. Intranasal co-administration of CDG/Ag induces immune response in lung and nasal-associated lymphoid tissue (NALT). Here, we will use TNFR1-/-TNFR2-/- and conditional STING-/- mice to dissect the in vivo mechanism of CDG/STING-induced adjuvant activity in lung and NALT. We will adopt both the model Ag OVA and pneumococcal surface protein A (PspA) Ag for our study. Dendritic cells (DC) play a central role in adjuvant activity. We hypothesize that CDG induces STING-dependent inflammatory signals that activate DC directly or indirectly to execute the adjuvant activity of CDG. Two specific Aims are proposed to carry out this objective. Aim 1. Determine how STING regulates CDG-induced adjuvant activity in DC. Aim 2. Determine how TNF-a regulates CDG-induced adjuvant activity. Currently, there is no vaccine formulation containing a mucosal adjuvant approved for human use. The knowledge generated by our studies can help advance the development of CDG as an efficacious mucosal vaccine adjuvant for human use.