Ndfip1 mediates peripheral tolerance to self and INAUGURAL ARTICLE exogenous antigen by inducing cell cycle exit in + responding CD4 T cells John A. Altina, Stephen R. Daleya, Jason Howittb, Helen J. Rickardsa, Alison K. Batkina, Keisuke Horikawaa, Simon J. Prasada, Keats A. Nelmsa, Sharad Kumarc, Lawren C. Wud, Seong-Seng Tanb, Matthew C. Cooka,1, and Christopher C. Goodnowa,1,2 aJohn Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia; bFlorey Neurosciences Institute, University of Melbourne, Parkville, VIC 3010, Australia; cCentre for Cancer Biology, University of South Australia, Adelaide, South Australia 5000, Australia; and dDepartment of Immunology, Genentech, South San Francisco, CA 94080 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2013. Contributed by Christopher C. Goodnow, December 17, 2013 (sent for review October 13, 2013) The NDFIP1 (neural precursor cell expressed, developmentally down- (11), rheumatoid arthritis (12), and multiple sclerosis (13). It regulated protein 4 family-interacting protein 1) adapter for the remains unclear which cellular mechanisms of tolerance are ubiquitin ligase ITCH is genetically linked to human allergic and auto- disrupted by these genetic variants to result in allergic and immune disease, but the cellular mechanism by which these proteins autoimmune disease. enable foreign and self-antigens to be tolerated is unresolved. Here, Ndfip1 and Itch were first revealed as important immune we use two unique mouse strains—an Ndfip1-YFP reporter and an regulators in mouse genetic studies. Homozygous inactivating Ndfip1-deficient strain—to show that Ndfip1 is progressively in- mutations in the Itchy strain cause dermatitis, lung mononuclear duced during T-cell differentiation and activation in vivo and that inflammation, lymphadenopathy with follicular hyperplasia, in- – its deficiency causes a cell-autonomous, Forkhead box P3-indepen- creased activated T cells (notably IL-4 producing Th2 cells), + dent failure of peripheral CD4 T-cell tolerance to self and exogenous expansion of B1b cells in the peritoneal cavity, and early death IMMUNOLOGY + antigen. In small cohorts of antigen-specific CD4 cells responding (5, 6, 14, 15). Although the murine pathology has often been in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit described as autoimmune because of its spontaneous de- cell cycle after one to five divisions and to abort Th2 effector differ- velopment, there is currently little direct evidence of T-cell au- toimmunity, and the predominant inflammation of skin and entiation, defining a step in peripheral tolerance that provides mucosal surfaces suggests an exaggerated response to innocuous insights into the phenomenon of T-cell anergy in vivo and is distinct environmental antigens. Indeed, elegant studies showed that Itch from the better understood process of Bcl2-interacting mediator Ndfip1 deficiency prevents high-zone tolerance in an experimental of cell death-mediated apoptosis. deficiency precipitated model of respiratory exposure to an egg protein allergen (16). autoimmune pancreatic destruction and diabetes; however, this An almost identical skin and lung inflammatory syndrome occurs depended on a further accumulation of nontolerant anti-self T cells in mice inheriting a homozygous gene-trap insertion that greatly from strong stimulation by exogenous tolerogen. These findings reduces Ndfip1 mRNA and protein (2). Although much progress illuminate a peripheral tolerance checkpoint that aborts T-cell has been made elucidating diverse biochemical functions of Itch clonal expansion against allergens and autoantigens and demon- strate how hypersensitive responses to environmental antigens may Significance trigger autoimmunity. Advances in organ transplantation and treatment of allergy immunological tolerance | allergy | T lymphocyte | Interleukin-4 | and autoimmune disease hinge upon harnessing a physiologi- Aire (Autoimmune Regulator) cal switch that allows T cells to decide between proliferating extensively or actively becoming tolerant. The experiments n healthy individuals, mature T cells in peripheral lymphoid presented here illuminate a critical element of this natural switch, Itissues proliferate and acquire effector functions in response to Ndfip1 (neural precursor cell expressed, developmentally down- antigens from pathogenic microbes but remain tolerant to self- regulated protein 4 family-interacting protein 1), a partner pro- antigens and innocuous environmental antigens. Defects in this tein of ubiquitin ligases induced during the first several divisions phenomenon of “peripheral T-cell tolerance” are thought to after T cells encounter antigen. They define the cellular action of contribute to the burden of autoimmune and allergic disease, but Ndfip1 in vivo, acting within dividing helper T cells that have there is only a fragmented understanding of its cellular basis, its responded to innocuous foreign or self-antigen that should connection to specific genetic circuits, and the interconnection normally be tolerated, to force their exit from cell cycle before between autoimmunity and hypersensitivity to exogenous anti- they have divided so many times that they acquire tissue-dam- gens (1). This problem is exemplified by the genetic circuit aging effector functions. encoding Ndfip1 [neural precursor cell expressed, developmentally down-regulated protein 4 (NEDD4) family-interacting protein 1], Author contributions: J.A.A., S.R.D., J.H., H.J.R., A.K.B., K.H., S.J.P., K.A.N., L.C.W., S.-S.T., M.C.C., and C.C.G. designed research; J.A.A., S.R.D., J.H., H.J.R., A.K.B., K.H., S.J.P., K.A.N., a transmembrane protein localized to the Golgi and intracellular and M.C.C. performed research; J.H., S.K., and S.-S.T. contributed new reagents/analytic vesicles that recruits and activates the HECT-type E3 ubiquitin tools; J.A.A., S.R.D., J.H., H.J.R., A.K.B., K.H., K.A.N., L.C.W., S.-S.T., M.C.C., and C.C.G. ligase Itch (2–7). Human genetic studies have associated NDFIP1 analyzed data; and J.A.A., S.R.D., M.C.C., and C.C.G. wrote the paper. and ITCH with allergic and autoimmune diseases. Inherited ITCH The authors declare no conflict of interest. deficiency results in asthma-like chronic lung disease with non- Freely available online through the PNAS open access option. fibrotic lymphocytic pneumonitis (90% cases) and organ-specific 1M.C.C. and C.C.G. contributed equally to this work. autoimmunity (60% cases) variably involving the thyroid, liver, 2To whom correspondence should be addressed. E-mail: [email protected]. intestine, or pancreatic islets (8). Inherited NDFIP1 polymorphisms This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are associated with inflammatory bowel disease (9, 10), asthma 1073/pnas.1322739111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322739111 PNAS | February 11, 2014 | vol. 111 | no. 6 | 2067–2074 Downloaded by guest on October 2, 2021 and Ndfip1 in many cell types (3, 17), the cellular basis for im- A BC mune dysregulation in their absence is unresolved, and their role C -58 nt in T-cell tolerance to self-antigens has yet to be examined. Exon 4 Exon 5 Exon 6 KO mutant Defects in several different cellular mechanisms for peripheral Wt: GTATG…….GG gt wild-type Mut: GTATG…….GG at -125 nt T-cell tolerance have been implicated in the inflammatory dis- -58 nt PY? ease caused by defects in the Itch-Ndfip1 genetic circuit. T-cell -125 nt PY2 Ndfip1 anergy is a mechanism defined initially in tissue culture that PY1 prevents initiation of T-cell proliferation when T cells are stim- D N ulated without a CD28 costimulus (18). Itch was required for 100 100 T-cell anergy in cultured cells rendered anergic by prolonged in 80 80 60 60 vitro treatment with ionomycin or harvested from TCR trans- 40 40 tubulin genic (Tg) mice 10 d after exposure to a high-tolerogenic dose of 20 20 foreign antigen. An intact Itch gene was correlated with di- 0 % surviving 0 % dermatitis-free 0 100 200 0 100 200 minished TCR signaling and proteolytic degradation of protein age (days) age (days) θ γ kru/kru +/+ , +/– kinase C (PKC)- , phospholipase C (PLC)- , JunB, and c-Jun Ndfip1(n=13)(n=13) Rag (n=13) kru/kru –/– proteins (16, 19). A role for Itch in nondegradative ubiquitina- Ndfip1(n=27)(n=27) Rag (n=27) tion of the TCR CD3 ζ subunit to inhibit its phosphorylation and the activation of Zap-70 has also been shown (20). Likewise, Fig. 1. Immune-mediated lethal inflammatory syndrome in mice with Ndfip1 deficiency causes JunB accumulation (2) and allows T a truncating Ndfip1 splice site mutation. (A) Schematic showing the location of the Ndfip1kru mutation within the Ndfip1 exon 5 splice donor sequence cells to make IL-2 for a sustained period in vitro without the and the resulting two aberrant splice products. (B) Schematic showing the need for CD28 costimulation (21). Itch-deficient T cells also κ α topology of the Ndfip1 protein and position of the truncating mutations. (C) display exaggerated NF- B signaling in response to TNF- , be- Western blot of primary T-cell lysates from wild-type, Ndfip1kru/kru (mutant), − − cause Itch forms a complex that recruits Tnfaip3 to terminate and Ndfip1 / (KO) mice, probed with an antibody raised to a conserved N- NF-κB signaling (22). Itch has also been implicated in ubiquiti- terminal peptide of Ndfip1, and then stripped and reprobed with antibody nation and degradation of Bcl-10, an adaptor protein with an to tubulin to assess loading. (D) Dermatitis and survival of Ndfip1kru/kru mice essential role in TCR and CD28 signaling to NF-κB (23). TCR- with normal or null Rag1 genes, aged to 250 d or until moribund necessi- activated T cells that lack Itch or Ndfip1 form a diminished tating the animal to be killed.