The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function This information is current as Yenkel Grinberg-Bleyer, Rachel Caron, John J. Seeley, of September 25, 2021. Nilushi S. De Silva, Christian W. Schindler, Matthew S. Hayden, Ulf Klein and Sankar Ghosh J Immunol 2018; 200:2362-2371; Prepublished online 19 February 2018; doi: 10.4049/jimmunol.1800042 Downloaded from http://www.jimmunol.org/content/200/7/2362 Supplementary http://www.jimmunol.org/content/suppl/2018/02/16/jimmunol.180004 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 50 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/200/7/2362.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Alternative NF-kB Pathway in Regulatory T Cell Homeostasis and Suppressive Function Yenkel Grinberg-Bleyer,* Rachel Caron,* John J. Seeley,* Nilushi S. De Silva,*,†,‡ Christian W. Schindler,* Matthew S. Hayden,*,1 Ulf Klein,*,†,‡,2 and Sankar Ghosh* CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-kB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-kB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Downloaded from Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-kB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity. The Journal of Immunology, 2018, 200: 2362–2371. http://www.jimmunol.org/ aintenance of immune homeostasis and tolerance is the constitutive expression of CD25 and the forkhead-box tran- achieved through multiple feedback mechanisms. scription factor Foxp3, which is required for their suppressive CD4+Foxp3+ regulatory T cells (Tregs) represent activity (2). Perturbations in the homeostasis and/or function of M + 5–15% of the CD4 T cell population and develop both in the these cells are associated with the development of autoimmune thymus (natural Treg [nTreg]) and the periphery (peripheral Treg diseases such as type 1 diabetes and rheumatoid arthritis (3). [pTreg]). They play a pivotal role in the control of innate and Depletion of Tregs in mice leads to a lethal multifocal inflam- adaptive immune responses (1), and are generally characterized by mation; moreover, mutations in the foxp3 locus are responsible for the Scurfy phenotype in mice and the IPEX (immunodysregula- by guest on September 25, 2021 *Department of Microbiology and Immunology, College of Physicians and Surgeons, tion, polyendocrinopathy, and enteropathy, X-linked) syndrome in Columbia University, New York, NY 10032; †Herbert Irving Comprehensive Cancer humans (4). Thus, Treg development, maintenance, and function Center, College of Physicians and Surgeons, Columbia University, New York, NY must be tightly regulated through various molecular mechanisms. 10032; and ‡Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 In recent years, several studies have demonstrated a crucial role 1Current address: Section of Dermatology, Department of Surgery, Dartmouth- for the NF-kB transcription factor in the development of Tregs and Hitchcock Medical Center, Lebanon, NH. the expression of Foxp3 (5–7). 2Current address: Section of Experimental Haematology, Leeds Institute of Cancer NF-kB signaling can be separated in two main pathways. The and Pathology, University of Leeds, Leeds, U.K. canonical pathway leads to the phosphorylation and degradation ORCIDs: 0000-0002-3515-8305 (Y.G.-B.); 0000-0003-1421-4256 (R.C.); 0000- of IkBa/b and consequent nuclear translocation of the NF-kB1 0002-4789-967X (U.K.). p50 protein bound to either c-Rel or p65, whereas the alternative Received for publication January 9, 2018. Accepted for publication January 31, 2018. pathway leads to activation of NF-kB–inducing kinase (NIK), This work was supported by grants from the National Institutes of Health (R01- which promotes processing of NF-kB2 from the full-length pre- AI068977) and from the Herbert Irving Cancer Center at Columbia University (to S.G.). Y.G.-B. was supported by a postdoctoral fellowship from the Cancer Research cursor protein p100 to the p52 form, which results in formation of Institute. transcriptionally active p52:RelB complexes (8, 9). The biological Y.G.-B., M.S.H., and S.G. wrote the paper. Y.G.-B. and S.G. conceived experiments. processes regulated by these two NF-kB pathways are distinct. Y.G.-B., R.C., and J.J.S. performed experiments. Y.G.-B., C.W.S., M.S.H., and S.G. The canonical p65 and c-Rel subunits are well-defined inducers of analyzed the data. N.S.D.S. and U.K. provided mice and reagents. S.G. secured funding. inflammation and have clear roles in the activation of B and T cells, whereas the alternative NF-kB RelB:p52 heterodimer has The sequences presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE108532) under primarily been implicated in lymphoid tissue organogenesis and accession number GSE108532. cell migration (9, 10). NF-kB2 and RelB are important regulators 2 2 2 2 2 2 Address correspondence and reprint requests to Dr. Sankar Ghosh, Department of immune tolerance, because NIK / , relb / , and nfkb2 / of Microbiology and Immunology, College of Physicians and Surgeons, Columbia mice develop spontaneous autoimmunity (reviewed in Ref. 11). University, 701 W 168th Street, HHSC 1204, New York, NY 10032. E-mail address: [email protected] Importantly, the two NF-kB pathways are triggered by different The online version of this article contains supplemental material. receptors and signaling pathways. Upon engagement of the TCR/ Abbreviations used in this article: BM, bone marrow; GSEA, Gene Set Enrichment CD28 Ag receptor, the canonical pathway is activated and NF-kB Analysis; KO, knockout; LN, lymph node; NIK, NF-kB–inducing kinase; nTreg, complexes containing c-Rel can regulate de novo expression of natural Treg; pLN, peripheral LN; pTreg, peripheral Treg; Tconv, pconventional Foxp3 in developing thymocytes, and therefore c-Rel–deficient CD4+ T; Tg, transgenic; Treg, regulatory T cell; WT, wild-type. animals exhibit fewer nTregs (5–7, 12–15). Recently, we and Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 others have demonstrated a central role for the NF-kB p65 subunit www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800042 The Journal of Immunology 2363 in the maintenance of mature Treg identity and in the prevention (Life Technologies) with 105 T cell–depleted, mitomycin C–treated wild- of autoimmunity (16, 17). In contrast, c-Rel, but not p65, is re- type (WT) splenocytes and 2.5 mg/ml anti-mCD3 (Bio X Cell), in the quired for the homeostasis of Tregs during antitumor responses presence of 10 ng/ml mIL-2 (PeproTech) and grading doses of human TGF-b1 (PeproTech), for 4 d at 37˚C. Cells were then stained for flow (18). These observations highlight canonical NF-kB signaling as a cytometry analysis. master regulator of Treg development and function, and demon- strate the discrete functions of individual NF-kB subunits in Treg- Suppression assays dependent immune tolerance. For in vitro assays, LN-derived CD45.1+ naive conventional CD4+ T The alternative NF-kB signaling pathway, by contrast, is largely (Tconv) cells were magnetically isolated (Miltenyi) and labeled with inactive during normal T cell homeostasis. In lymphocytes, NF- CellTrace Violet Proliferation Tracker (CTV; Life Technologies). They were cultured with T cell–depleted, mitomycin C–treated WT splenocytes kB2 resides predominantly in the cytoplasm in its unprocessed and 2.5 mg/ml anti-mCD3, in the presence or not of FACS-sorted WT or p100 form, where it is believed to function as an inhibitor of other nfkb22/2 CD4+YFP+ Tregs. Proliferation of CD4+CD45.1+ T cells was NF-kB proteins (9). However, activation of a subset of TNFR assessed by FACS at day 4. The percentage of suppression was calculated 5 + family members expressed in lymphocytes, including OX40, as described. For in vivo assays, 4 3 10 naive CD45.1 Tconv cells were isolated as described earlier and transferred with or without 1 3 105 Tregs CD40, or LT-bR, leads to activation of the alternative pathway. 2/2 to the retro-orbital sinus of 6- to 9-wk-old RAG1 mice.