In response to Luteijn et al.: Concerns regarding cGAMP uptake assay and evidence that SLC19A1 is not the major cGAMP importer in human PBMCs

We previously reported that SLC19A1 is an importer of the immunotransmitter 23-cyclic-GMP-AMP (cGAMP)1 by performing a genome wide screen in U937 cells. Soon after, Lutejin et al. reported similar findings by conducting a screen in THP-1 cells2. While the conclusions of these two studies largely overlap, we arrived at significantly different conclusions regarding how broadly SLC19A1 is used by different cell types. Our study suggests that in addition to SLC19A1, many cultured and primary cell types use alternative, unidentified transporters to import cGAMP and other cyclic dinucleotides (CDNs). This conclusion was based on our findings that inhibition of SLC19A1 did not significantly reduce extracellular cGAMP signaling in multiple cell types, including primary CD14+peripheral blood mononuclear cells (PBMCs) from most donors. In contrast, Luteijn et al. concluded that SLC19A1 is the major CDN importer in humans, largely based on their use of a radiolabeled [32P] cGAMP uptake assay. Using this assay, they showed that inhibition of SLC19A1 abolishes [32P] uptake in total PBMCs. However, they did not test whether inhibition of SLC19A1 affects extracellular cGAMP signaling in these cells. Here, we highlight an important issue with the [32P] cGAMP uptake assay used by Luteijn et al. and demonstrate that measuring extracellular cGAMP signaling through the STING pathway is currently the best method for evaluating cGAMP import. We also show that inhibition of SLC19A1 has no effect on extracellular cGAMP signaling in total PBMCs, confirming that this cell type relies on other transport mechanisms for cGAMP import.