A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans

Abstract

Abstract A20 is a ubiquitously expressed, inducible ubiquitin-editing enzyme that restrains NF-kB-dependent inflammatory signaling throughout the body. In T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-kB and c-Jun partner AP-1. Utilizing WT and A20KO Jurkat T cells, we demonstrate A20 is required to negatively regulate both NF-kB and JNK/AP-1 signaling. By employing a novel set of A20 mutant constructs with NF-kB and AP-1 driven reporter systems in Jurkat transfectants, we discovered the ZnF7 domain is essential for CBM localization and signal modulation. We confirmed these findings in CRISPR-mediated A20 knockout human primary effector T cells, demonstrating sustained TCR-induced NF-kB and JNK signaling, enhanced upregulation of activation markers, and increased cytokine secretion associated with a skewed Th9/Th17 phenotype. Moreover, loss of A20 altered apoptosis sensitivity in primary human T cells, resulting in decreased restimulation induced cell death (RICD) and increased cytokine withdrawal induced death (CWID). These findings further elucidate the mechanism of A20-dependent regulation of both NF-kB and c-Jun in T cells, and highlight the functional importance of A20 in maintaining adaptive immune homeostasis.