CRISPR/Cas9 Screens Reveal Multiple Layers of B Cell CD40 Regulation

Abstract

CD40 has major roles in B-cell development, activation and germinal center responses. CD40 hypoactivity causes immunodeficiency, whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B-cell autonomous CD40 regulators, we performed CRISPR/Cas9 genome-wide screens. These found CD40 pathway components and revealed mechanisms strongly influencing CD40 responses. At the transcriptional level, the ubiquitin ligase FBXO11 supported CD40 abundance by targeting the repressor CTBP1. FBXO11 knockout decreased primary B-cell CD40 expression and impaired class-switch recombination. Frequent lymphoma monoallelic FBXO11 mutations may therefore balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controlled exon splicing critical for CD40 protein expression, while the N6-adenosine methyltransferase WTAP controlled CD40 message abundance. At the protein level, ESCRT negatively-regulated activated CD40 levels while the negative feedback phosphatase DUSP10 downmodulated MAPK. These results reveal multiple layers of CD40 regulation, serve as a resource for future studies of this key co-receptor, and highlight potential therapeutic targets. CD40 has major roles in B-cell development, activation and germinal center responses. CD40 hypoactivity causes immunodeficiency, whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B-cell autonomous CD40 regulators, we performed CRISPR/Cas9 genome-wide screens. These found CD40 pathway components and revealed mechanisms strongly influencing CD40 responses. At the transcriptional level, the ubiquitin ligase FBXO11 supported CD40 abundance by targeting the repressor CTBP1. FBXO11 knockout decreased primary B-cell CD40 expression and impaired class-switch recombination. Frequent lymphoma monoallelic FBXO11 mutations may therefore balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controlled exon splicing critical for CD40 protein expression, while the N6-adenosine methyltransferase WTAP controlled CD40 message abundance. At the protein level, ESCRT negatively-regulated activated CD40 levels while the negative feedback phosphatase DUSP10 downmodulated MAPK. These results reveal multiple layers of CD40 regulation, serve as a resource for future studies of this key co-receptor, and highlight potential therapeutic targets.

Keywords: B cell activation, NF-kappaB, MAP kinase, CRISPR screen, TNF receptor superfamily, humoral immunity, immunodeficiency, N6-Methyladenosine, ESCRT, germinal center