PROJECT ABSTRACT One of the most important functions of innate immune receptors is the recognition of nucleic acids. Two key DNA binding receptors are the AIM2 (absent in melanoma) inflammasome and the cGAS (cyclic GMP-AMP synthase) protein. AIM2 is a member of the IFN-inducible HIN-200 family which is comprised of an N-terminal (pyrin) PYD and a C-terminal HIN-200 domain. Upon binding of DNA to the HIN-200 domain, the PYD is released to interact with the common inflammasome adaptor molecule, ASC (apoptoic speck containing protein with a CARD). This leads to the recruitment and activation of caspase-1, and the cleavage and maturation of IL-1, IL- 18 and the pyroptotic executioner, gasdermin D (GSDMD). AIM2 mediates inflammasome response to a wide range of bacterial, viral and self-derived DNA in a sequence-independent but length-dependent fashion. cGAS is a cytosolic enzyme that synthesizes a cyclic dinucleotide, cGAMP, from ATP and GTP. cGAMP in turn is recognized by the STING (stimulator of interferon genes) innate immune receptor, leading to the downstream activation of TBK1, NFB and IRF3 which cumulate in the transcriptional activation of type I interferon and numerous inflammatory cytokines. While much is known about the functional roles of these two molecules, critical knowledge remains missing. For example, limited information is available on the structure of DNA that is recognized by these molecules in myeloid cells. New preliminary findings lead us to hypothesize that the secondary structure of nucleic acids impacts their recognition and likely functional outcomes. Aim 1 will test this hypothesis. Another critical knowledge that is understudied centers on the roles of these innate immune receptors in adaptive immune cells. Innate immune receptors have been primarily studied in myeloid cells, however an analysis of their expression indicates that many are well expressed by adaptive T and B lymphocytes. We have recently shown that both AIM2 and STING play prominent and surprising roles in T and B cells. We find that AIM2 promotes T regulatory (Treg) cells in vitro and in vivo and this is due to the association of AIM2 with the RACK1 kinase and PP2a phosphatase complex, which then blocks Akt activation. We also unexpectedly found that STING induces an IL-35/IL-10 phenotype in B cells with markers and functions that are consistent with B regulatory cells. The relevance of these innate immune receptors in lymphocytes will be the focus of this proposal.