Resolving mechanisms of immune‐mediated disease in primary CD4 T cells

• Published in: EMBO molecular medicine 2020-05, Vol.12 (5), p.e12112-n/a
• Main Authors: Bourges, Christophe, Groff, Abigail F, Burren, Oliver S, Gerhardinger, Chiara, Mattioli, Kaia, Hutchinson, Anna, Hu, Theodore, Anand, Tanmay, Epping, Madeline W, Wallace, Chris, Smith, Kenneth GC, Rinn, John L, Lee, James C
• Format: Article
• Language: English
• Subjects: Autoimmune diseases; Autoimmunity; Biology; CD4 antigen; CD4 T cells; CD4-Positive T-Lymphocytes; Cell activation; Disease; EMBO10; EMBO16; EMBO19; Gene loci; Genomes; GWAS; Haplotypes; Humans; Linkage disequilibrium; Lymphocytes; Lymphocytes T; MPRA; Multiple sclerosis; NF-kappa B; Polymorphism, Single Nucleotide; Single-nucleotide polymorphism; super‐enhancer; TNFAIP3
• ISSN: 1757-4676; 1757-4684
• DOI: 10.15252/emmm.202012112

Deriving mechanisms of immune‐mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by strong linkage disequilibrium. To determine whether causal variants could be identified from their functional effects, we adapted a massively parallel reporter assay for use in primary CD4 T cells, the cell type whose regulatory DNA is most enriched for immune‐mediated disease SNPs. This enabled the effects of candidate SNPs to be examined in a relevant cellular context and generated testable hypotheses into disease mechanisms. To illustrate the power of this approach, we investigated a locus that has been linked to six immune‐mediated diseases but cannot be fine‐mapped. By studying the lead expression‐modulating SNP, we uncovered an NF‐κB‐driven regulatory circuit which constrains T‐cell activation through the dynamic formation of a super‐enhancer that upregulates TNFAIP3 (A20), a key NF‐κB inhibitor. In activated T cells, this feedback circuit is disrupted—and super‐enhancer formation prevented—by the risk variant at the lead SNP, leading to unrestrained T‐cell activation via a molecular mechanism that appears to broadly predispose to human autoimmunity. Synopsis Little progress has been made in resolving causal SNPs, genes and disease mechanisms at GWAS loci. An adapted massively‐parallel reporter assay (MPRA) allows to study immune‐mediated disease loci in CD4 T cells, the cell‐type whose regulatory DNA is most highly enriched for disease‐associated SNPs. Adapted MPRA identifies putative causal SNPs based on their functional effects within primary CD4 T cells—key effectors of immune‐mediated disease. These effects differ from those detected in the Jurkat cell‐line, reinforcing the importance of an appropriate cellular context in disease‐related studies. The results provide a focus for mechanistic studies to resolve the downstream consequences of expression‐modulating variants at multiple loci. At a gene‐desert linked to multiple diseases, the lead MPRA SNP is shown to abrogate NF‐κB binding, disrupt super‐enhancer formation, and reduce TNFAIP3 expression, leading to unrestrained T cell‐driven inflammation. This provides mechanistic insights into disease biology at a locus that cannot be fine‐mapped and illustrates the potential of this method to uncover genetic mechanisms of immune‐mediated disease. Graphical Abstract Little progress has been made in resolving causal SNPs, genes and disease