Identification of Plasmacytoid Dendritic Cell Regulators

SUMMARY Type I interferons (IFN-I) are essential in the defense against viral infection and cancers. However, aberrant, or excessive IFN-I production can be pathogenic and lead to the development of autoimmune diseases. Plasmacytoid dendritic cells (pDCs) produce more IFN-I than any other cell type and are thus critical to establish systemic defense against viral pathogens. Conversely, pDC dysfunction is strongly associated with many interferonopathies. We and others have found that although pDCs initially produce exceptional amounts of IFN-I following a viral infection, they lose their capacity to produce these cytokines, becoming “exhausted”, a few days later. This phenomenon, which is conserved in mice and humans, provides the virus an opportunity to persist and exposes the host to increased risk of secondary infections. pDCs with an exhausted phenotype have also been reported in many tumors. However, the mechanisms underlying pDC IFN-I production in general as well as pDC exhaustion are still not well understood. Although many studies have identified individual regulators of pDC IFN-I production, such as the transcription factor IRF7, to date no genome-wide investigation to identify regulators of pDC function has been performed. This is likely because pDCs are rare and short lived, and the available pDC-cell lines are suboptimal. We have now generated an improved pDC cell line (4C1), which produces high levels of IFNα and IFNβ, both of which are dependent on IRF7 and toll-like receptor (TLR). We have also engineered this new cell line with an IFN-I reporter and constitutive Cas9 expression to allow for genetic screening of pDC IFN-I regulators. Our preliminary data indicate that this reporter-pDC cell line can reliably measure IFN-I production after stimulation and can be genetically manipulated in a straightforward manner. Furthermore, we have developed a protocol for inducing exhaustion in this reporter-pDC cell line, which recapitulates the exhaustion gene signature and the cell-intrinsic TLR7 dependency that we reported during in vivo pDC exhaustion. Combining our new tools along with the Genome-Scale CRISPR Knock-Out (GeCKO) library, we propose the first genome-wide screen for regulators of both human pDC function and exhaustion. Furthermore, using protocols well established in our laboratory for the manipulation of human primary pDCs, we propose to verify and further characterize the top regulators selected from the screen in pDCs from healthy volunteers. Finally, we will validate these results using in vivo mouse models of viral infection. Our proposed work will provide the first genome-scale analysis of IFN-I regulation in pDCs, and give us exceptional and novel insights into the mechanisms that regulate IFN-I production and subsequent exhaustion in this unique and critical innate cell. These insights can then be leveraged to design therapies which are targeted to modulate IFN-I production in multiple human diseases.