SLC19A1 is a cyclic dinucleotide transporter

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage. Cytosolic DNA triggers immune responses by activating the cGAS/STING pathway. The binding of DNA to the cytosolic enzyme cGAMP synthase (cGAS), activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic[G(29,59)pA(3959)] (2939-cGAMP). 2939-cGAMP, a cyclic dinucleotide (CDN), activates the protein 9stimulator of interferon genes9 (STING), which in turn activates the transcription factors IRF3 and NF-κB promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2939-cGAMP and other CDNs, including CDNs produced by bacteria and synthetic CDNs used in cancer immunotherapy, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR interference screen to identify the reduced folate carrier SLC19A1 as the major CDN transporter for uptake of synthetic and naturally occurring CDNs. CDN uptake and functional responses are inhibited by depleting SLC19A1 from cells and enhanced by overexpressing SLC19A1. In both cell lines and primary cells ex vivo, CDN uptake is inhibited competitively by folate and blocked by the SLC19A1 inhibitor sulfasalazine, a medication approved for the treatment of inflammatory diseases. The identification of SLC19A1 as the major transporter of CDNs into cells has far reaching implications for the immunotherapeutic treatment of cancer, transport of 2939-cGAMP from tumor cells to other immune cells to trigger the anti-tumor immune response, host responsiveness to CDN-producing pathogenic microorganisms, and potentially in certain inflammatory diseases.