Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3

Abstract

Abstract Although prior research implicated c-Jun N-terminal kinase (JNK) in helper CD4 +T cell differentiation, mechanisms governing JNK signaling and function in human lymphocytes remain nebulous. CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor (AgR) engagement to transcriptional programs necessary for effector cell survival, proliferation, and differentiation. In addition to NF-κB, CARD11 is also required for AgR-induced mTORC1 and JNK2 signaling. We sought to define the impact of dominant interfering (DI) CARD11 variants on JNK signaling and elucidate transcriptomic changes governed by CARD11-dependent JNK activation in T cells. We transfected WT and CARD11 knockout (KO) Jurkat T cells with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling and performed RNA sequencing on mitogen-activated WT and CARD11 KO Jurkat T cells ± JNK inhibition. Our results revealed that AgR-dependent JNK1 andJNK2 activation are CARD11-dependent. Additionally, heterozygous DI CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, mirroring the extent of impaired NF-κB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4 +T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. Our novel findings suggest that defects in CARD11-dependent JNK signaling in CD4 +T cells may contribute to severe allergic disease noted in CADINS patients, unveiling a new potential therapeutic target. Supported by funding from the Jeffrey Modell Specific Defect Research Program (ALS) and the Henry M. Jackson Foundation for the Advancement of Military Medicine (BMB).