Averil Ma

Macrophages play critical roles in regulating host responses to microbial pathogens and other forms of tissue stress and can acquire pro-inflammatory or tissue reparative phenotypes. A20, or TNFAIP3, is a potent regulator of innate immune cell functions and is extensively linked to human inflammatory and autoimmune diseases. We now find that A20 is a powerful regulator of both glycolytic and mitochondrial respiration in macrophages. Differentiated macrophages that are acutely rendered A20 deficient exhibit increased glycolytic activity and markedly decreased mitochondrial respiration after LPS stimulation. These cells are unable to repolarize from an M1-like to M2-like phenotype. Compromised mitochondrial oxygen consumption in A20 deficient macrophages is caused by increased nitric oxide production. Inhibition or genetic ablation of inducible nitric oxide synthase (iNOS) restores mitochondrial oxidative phosphorylation and lactate production in these cells. These metabolic perturbations occur independently of exaggerated cytokine production and despite robust production of IL-10. Therefore, A20 prevents Warburg-like aerobic glycolysis and restores macrophage homeostasis.

T cell exhaustion limits the efficacy of cancer immunotherapies. Here, we performed genome-wide loss-of-function screening in repetitively stimulated human T cells and identified the mulitfunctional ubiquitin-modifying protein A20/TNFAIP3 as a major negative regulator of exhausted T cell persistence. Protein large language modeling, deep base-editing mutagenesis, and studies in immunocompetent mice with domain-specific inactivating mutations revealed A20’s non-enzymatic M1 ubiquitin-binding zinc finger 7 (A20 ^ZF7 ) motif as critical to suppression of anti-tumor immunity. A20 ^ZF7 -deficient CD8 ⁺ tumor-infiltrating lymphocytes (TILs) resisted terminal exhaustion and circumvented an unappreciated mechanism restraining perforin degranulation in terminally exhausted cells. Human chimeric antigen receptor (CAR)-T cells engineered via base-editing to inactivate A20 ^ZF7 via a single missense mutation also resisted exhaustion, secreted more perforin and robustly suppressed cancer in vivo . These studies pinpoint A20 ^ZF7 as a novel T cell checkpoint and reveal precision base-editing of missense mutations as an effective approach to enhance CAR-T cell therapy.

A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn’s disease and celiac disease in humans. We now find that mice expressing point mutations in A20’s M1-ubiquitin–binding zinc finger 7 (ZF7) motif spontaneously develop proximal enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveals expansion of Th17 cells and exuberant expression of IL-17A and IL-22 in intestinal lamina propria of A20 ^ZF7 mice. While deletion of IL-17A from A20 ^ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. Colonization of adult germ-free mice with microbiota from adult WT specific pathogen–free mice drives duodenal IL-22 expression and duodenitis. A20 ^ZF7/ZF7 Th17 cells autonomously express more RORγt and IL-22 after differentiation in vitro. ATAC sequencing identified an enhancer region upstream of the Il22 gene, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription in A20 ^ZF7/ZF7 T cells. Acute inhibition of RORγt normalized histone acetylation at this enhancer. Finally, CRISPR/Cas9–mediated ablation of A20 ^ZF7 in human T cells increases RORγt expression and IL22 transcription. These studies link A20’s M1-ubiquitin binding function with RORγt expression, expansion of Th17 cells, and epigenetic activation of IL-22–driven enteritis.

Polymorphisms in the TNFAIP3 locus encoding the A20 protein are strongly associated with psoriatic skin and joint disease. Reduced A20 expression, driven by both genetic and epigenetic factors, underscores its critical role as a negative regulator of psoriatic disease (PsD). Our recent study using a germline knockin mouse model harboring a mutation in A20’s seventh zinc finger, which impairs A20 binding to linear (M1) ubiquitin, revealed a spontaneous phenotype resembling psoriatic arthritis. These mice demonstrated sustained nuclear factor–κB signaling in response to transient tumor necrosis factor stimulation, leading to inappropriate transcription of mid- and late-response inflammatory genes. These findings highlight the role of dysregulated innate immune-signaling kinetics as a potential driver of PsD pathogenesis. These findings, together with distinct inflammatory mouse models resulting from temporally extended inflammatory gene activation, highlight the role of dysregulated innate immune-signaling kinetics as a potential driver of tissue inflammation with relevance to psoriatic skin and joint disease. This newly developed mouse model of PsD was presented at the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) 2024 annual meeting.

A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn’s disease and celiac disease in humans. We now find that mice expressing point mutations in A20’s M1 ubiquitin binding motif (ZF7) spontaneously develop proximate enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveal expansion of TH17/22 cells and aberrant expression of IL-17A and IL-22 in intestinal lamina propria of A20 ^ZF7 mice. While deletion of IL-17A from A20 ^ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. A20 ^ZF7/ZF7 TH17/22 cells autonomously express more RORψt and IL-22 after differentiation in vitro . ATAC sequencing identified an enhancer region upstream of the Il22 gene in A20 ^ZF7/ZF7 T cells, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription. Finally, CRISPR/Cas9-mediated ablation of A20 ^ZF7 in human T cells increases RORψt expression and IL22 transcription. These studies link A20’s M1 ubiquitin binding function with RORψt expression, epigenetic activation of TH17/22 cells, and IL-22 driven enteritis.

Acute myeloid leukemia (AML) is a deadly hematopoietic malignancy. Although many patients achieve complete remission with standard induction therapy, a combination of cytarabine and anthracycline, ~40% of patients have induction failure. These refractory patients pose a treatment challenge, as they do not respond to salvage therapy or allogeneic stem cell transplant. Herein, we show that AML patients who experience induction failure have elevated expression of the NF-κB target gene tumor necrosis factor alpha-induced protein-3 (TNFAIP3/A20) and impaired necroptotic cell death. A20^High AML are resistant to anthracyclines, while A20^Low AML are sensitive. Loss of A20 in AML restores sensitivity to anthracycline treatment by inducing necroptosis. Moreover, A20 prevents necroptosis in AML by targeting the necroptosis effector RIPK1, and anthracycline-induced necroptosis is abrogated in A20^High AML. These findings suggest that NF-κB-driven A20 overexpression plays a role in failed chemotherapy induction and highlights the potential of targeting an alternative cell death pathway in AML.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 ( Tnfaip3 ) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.