CRISPR screen finds Prostaglandin E receptor 2 modulates activation of dendritic cells and cross-presentation of cell-associated antigens from fibroblasts

Abstract

The sensing of pathogenic stimuli by dendritic cells (DCs) promotes antigen processing for cross-presentation, whereby the activation of DCs is followed by their migration to lymph nodes and spleen, where the cross-presented antigens can prime naïve T-cells for an adaptive immune response. DCs can also cross-present antigens in the absence of infection, but endogenous signals that promote antigen processing and presentation remain poorly understood. We observed that DCs efficiently cross-present cell-associated antigens from fibroblasts (3T3), even in the absence of pathogenic stimulus. We hypothesized that signals that promote DC activation might also promote processing of cell-associated antigens for presentation. In order to determine the molecular components responsible for DC activation, we performed a CRISPR knockout screen targeting a total of 704 known or predicted regulators involved in sensing of pathogens/danger signals. DCs were co-cultured with 3T3 and sorted into two populations for high and low expression of activation markers on DCs and the guides enriched were determined by sequencing. Prostaglandin E receptor 2 (PTGER2) was found to be the major hit. The results were validated in cDC1-like cell line, MutuDC, where direct activation of PTGER2 using its ligand prostaglandin E2 (PGE2) resulted in increased expression of activation markers (CD83 and CD86). Inhibition of the PGE2 production or of its receptor using small molecule inhibitors, resulted in significantly reduced activation of dendritic cells when co-cultured with 3T3 cells. The DC activation via PTGER2 was found to be mediated via cAMP production upon adenylyl cyclase activation. In addition, the inhibition of PTGER2 and PGE2 production via COX1/COX2 also resulted in reduced cross-presentation of cell-associated antigens. CRISPR screen found PTGER2 as the molecular component in dendritic cells that can regulate both DC activation and cross-presentation of cell-associated antigens in the absence of infection.