Tu112 - A Genome-wide CRISPR Screening Identifies Targets That Drive Tolerogenic Dendritic Cells

Abstract Text: Tolerogenic dendritic cells (TolDCs) play a crucial role in maintaining immune tolerance and are promising candidates for treating autoimmune diseases. Despite its importance in the clinic, the key molecular pathways behind TolDC-mediated tolerance remain unclear. These cells are typically characterized by a functional status rather than a specific lineage marker, creating a challenge for identification of key modulators of tolerance. To overcome this, we identified a phenotypic biomarker representing TolDCs' functional status, allowing us to perform a genome-wide pooled CRISPR screen to discover new therapeutic targets that promote TolDC function. First, we confirmed that CD86 is consistently and significantly downregulated upon all tested conditions under treatment with different tolerizing agents (dexamethasone, vitamin D3 and rapamycin) in bone marrow-derived dendritic cells and human monocyte-derived dendritic cells. Furthermore, by using CD86 blocking Abs and CRISPR KO, we demonstrated the dependency of TolDC on CD86 inactivation to induce tolerance by suppressing T cell proliferation and activation, highlighting CD86 as a key screening read-out for assessing tolerogenic potential of DCs. Finally, through a genome-wide CRISPR-Cas9 screen, we sorted cells based on CD86 expression levels, which enabled us to identify and validate several genes that enhance the TolDC phenotype. We also uncovered critical molecular pathways necessary for producing highly effective TolDCs. Together, these projects are aiming at restoring immune tolerance in patients with IMIDs in line with our overall goal to induce the next wave of therapies that drive a cure.