Genome-wide CRISPER screen in Dendritic cells to identify new regulatory genes that control immune suppression

Dendritic cells (DCs) play major role in host defense, presenting antigen to T cells and controlling T cells activation. One of the critical signals that enables T cell activation are mediated by B7 receptors, which contain two important co-stimulating receptors, B7-1(CD80) and B7-2(CD86) expressed by DCs. Interleukin 10 (IL-10), an anti-inflammatory cytokine, suppresses DCs function and reduces expression level of CD86 on cell surface. To find genes that mediate IL-10 suppressive effect in DCs, we performed a genome-wide pooled CRISPR-Cas9 screen in bone marrow differentiated dendritic cells. We aimed to identify genes that regulate expression of CD86 by co-treating IL10 and bacterial lipopolysaccharide (LPS). The 5% low/high CD86 expressed cells have been sorted and deep sequenced. A set of genes were found as potential negative regulator of CD86. To validate the screen results, we currently target individual genes. We validated two ubiquitin enzymes Usp18 and Uba3 that increase the expression of CD86, suggesting that these genes were negative regulator of CD86. Uba3 is activating enzyme 1 of NEDD8, a ubiquitin-like protein, which regulates cell division, signaling and embryogenesis. Usp18, a deubiquitinating protease enzymes, is de-conjugating protease of ISG15 and has been reported to function in innate immune system. Our result suggests Uba3 and Usp18 play important role in regulating the level of CD86 in DCs and we are investigating the molecular mechanism.