A20 is an anti-inflammatory protein linked to multiple human diseases, however the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We now find that A20 deficient T cells and fibroblasts are susceptible to caspase independent and RIPK3 dependent necroptosis. Global RIPK3 deficiency significantly rescues the survival of A20 deficient mice. A20 deficient cells exhibit exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 undergoes physiological ubiquitination at lysine 5 (K5), and this ubiquitination event supports the formation of RIPK1-RIPK3 complexes. The catalytic cysteine of A20’s deubiquitinating motif is required for inhibiting RIPK3 ubiquitination and RIPK1-RIPK3 complex formation. These studies link A20 and RIPK3 ubiquitination to necroptotic cell death, and suggest new mechanisms by which A20 may prevent inflammatory disease.

Dendritic epidermal T cells (DETCs) are a highly specialized population of γδ T cells that resides in the murine skin and participates in wound healing and tumor surveillance. Despite the expression of other stimulatory receptors on these cells, mechanisms involving activation have focused primarily on the invariant Vγ3-Vδ1 TCR expressed by DETCs. All DETCs also express the activating NKG2D receptor, but the role of NKG2D in DETC activation remains unclear, as does the identity of NKG2D ligands that are functionally expressed in the skin. In this study, we document the cloning of an NKG2D ligand H60c that is expressed specifically in the skin and in cultured keratinocytes and demonstrate its role in the activation of DETCs and NK cells. The ligand is unique among NKG2D ligands in being up-regulated in cultured keratinocytes, and its interaction with NKG2D is essential for DETC activation. Importantly, it is shown that engagement of NKG2D is not sufficient to activate DETCs, but instead provides a costimulatory signal that is nevertheless essential for activating DETCs in response to stimulation with keratinocytes.

A20 () and ABIN-1 () are candidate susceptibility genes for inflammatory bowel disease and other autoimmune or inflammatory diseases, but it is unclear how these proteins interact in vivo to prevent disease. Here we show that intestinal epithelial cell (IEC)-specific deletion of either A20 or ABIN-1 alone leads to negligible IEC loss, whereas simultaneous deletion of both A20 and ABIN-1 leads to rapid IEC death and mouse lethality. Deletion of both A20 and ABIN-1 from enteroids causes spontaneous cell death in the absence of microbes or hematopoietic cells. Studies with enteroids reveal that A20 and ABIN-1 synergistically restrict death by inhibiting TNF-induced caspase 8 activation and RIPK1 kinase activity. Inhibition of RIPK1 kinase activity alone, or caspase inhibition combined with RIPK3 deletion, abrogates IEC death by blocking both apoptosis and necroptosis in A20 and ABIN-1 double-deficient cells. These data show that the disease susceptibility proteins A20 and ABIN-1 synergistically prevent intestinal inflammation by restricting IEC death and preserving tissue integrity.

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here we find that mice expressing three distinct targeted mutations of A20’s ZF7 ubiquitin binding motif uniformly developed digit arthritis that shares features with psoriatic arthritis, while mice expressing point mutations in A20’s OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20 ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor (TNF) and interleukin 17A, and persisted in germ-free conditions. A20 ZF7 cells exhibited prolonged IKK kinase activity that drove exaggerated transcription of late-phase NF-κB-response genes in vitro and in pre-diseased mouse paws in vivo . In addition, mice expressing double-mutant A20 proteins in A20’s ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20’s ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.

SUMMARY During immune responses, naïve T cells transition from small quiescent cells to rapidly cycling cells. We have found that T cells lacking TAX1BP1 exhibit delays in growth of cell size and cell cycling. TAX1BP1-deficient T cells exited G0 but stalled in S phase, due to both bioenergetic and biosynthetic defects. These defects were due to deficiencies in mTOR complex formation and activation. These mTOR defects in turn resulted from defective autophagy induction. TAX1BP1 binding of LC3 and GABARAP via its LC3-interacting region (LIR), but not its ubiquitin-binding domain, supported T cell proliferation. Supplementation of TAX1BP1-deficient T cells with metabolically active L-cysteine rescued mTOR activation and proliferation but not autophagy. These studies reveal that TAX1BP1 drives a specialized form of autophagy, providing critical amino acids that activate mTOR and enable the metabolic transition of activated T cells.