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IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4 + T Cells

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IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4 + T Cells IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4 + T Cells This information is current as Michael L. Ridley, Veerle Fleskens, Ceri A. Roberts, of September 28, 2021. Sylvine Lalnunhlimi, Aldana Alnesf, Aoife M. O'Byrne, Kathryn J. A. Steel, Giovanni A. M. Povoleri, Jonathan Sumner, Paul Lavender and Leonie S. Taams J Immunol published online 22 April 2020 http://www.jimmunol.org/content/early/2020/04/17/jimmun Downloaded from ol.1901283 Supplementary http://www.jimmunol.org/content/suppl/2020/04/17/jimmunol.190128 Material 3.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • 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 Author Choice Freely available online through The Journal of Immunology Author Choice option 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 © 2020 The Authors All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 22, 2020, doi:10.4049/jimmunol.1901283 The Journal of Immunology IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4+ T Cells Michael L. Ridley,*,1 Veerle Fleskens,*,1 Ceri A. Roberts,* Sylvine Lalnunhlimi,* Aldana Alnesf,* Aoife M. O’Byrne,* Kathryn J. A. Steel,* Giovanni A. M. Povoleri,* Jonathan Sumner,† Paul Lavender,‡ and Leonie S. Taams* The expression of anti-inflammatory IL-10 by CD4+ T cells is indispensable for immune homeostasis, as it allows T cells to moderate their effector function. We previously showed that TNF-a blockade during T cell stimulation in CD4+ T cell/monocyte cocultures resulted in maintenance of IL-10–producing T cells and identified IKZF3 as a putative regulator of IL-10. In this study, we tested the hypothesis that IKZF3 is a transcriptional regulator of IL-10 using a human CD4+ T cell–only culture system. IL-10+ CD4+ T cells expressed the highest levels of IKZF3 both ex vivo and after activation compared with IL-10–CD4+ T cells. Pharmacological targeting of IKZF3 with the drug lenalidomide showed that IKZF3 is required for anti-CD3/CD28 mAb– Downloaded from mediated induction of IL-10 but is dispensable for ex vivo IL-10 expression. However, overexpression of IKZF3 was unable to upregulate IL-10 at the mRNA or protein level in CD4+ T cells and did not drive the transcription of the IL10 promoter or putative local enhancer constructs. Collectively, these data indicate that IKZF3 is associated with but not sufficient for IL-10 expression in CD4+ T cells. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/ he production of IL-10 by CD4+ T cells is key for the control deletions of Il10 lead to a pronounced inflammation in the colonic of effector function in response to immune challenge (1–3). mucosa in response to commensal gut bacteria (1). T Even in the absence of pathogens, CD4+ T cell–specific IKZF3 (encoding for the protein Aiolos) is a member of the Ikaros Zinc finger family of transcription factors (4). This gene is expressed by various immune cell types and has been implicated *Centre for Inflammation Biology and Cancer Immunology, Department of Inflam- in the function of multiple Th subsets (5, 6) as well as in con- mation Biology, School of Immunology and Microbial Sciences, King’s College † trolling CD4/CD8 fate decision in the thymus (7). The expression London, London SE1 1UL, United Kingdom; Department of Infectious Diseases, + School of Immunology and Microbial Sciences, King’s College London, Guy’s of IKZF3 in IL-17–producing CD4 T cells (Th17 cells) is as- Hospital, London SE1 9RT, United Kingdom; and ‡MRC and Asthma UK Centre sociated with a “nonpathogenic” signature, which includes in- by guest on September 28, 2021 in Allergic Mechanisms of Asthma, Peter Gorer Department of Immunobiology, creased IL-10 production (6, 8). IKZF3 has also been shown to School of Immunology and Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom interact with known regulators of IL10 expression, including its 1M.L.R. and V.F. contributed equally. most closely related family member IKZF1 (encoding Ikaros) (4) ORCIDs: 0000-0002-3953-2384 (M.L.R.); 0000-0002-7900-667X (C.A.R.); 0000- whichhasbeenshowninmicetodirectly affect the expression of 0001-8185-2501 (A.M.O.); 0000-0003-0342-651X (G.A.M.P.); 0000-0001-8710- Il10 (9). 0263 (P.L.); 0000-0002-9337-7194 (L.S.T.). Whereas IKZF3 has been suggested to act as a transcriptional Received for publication October 24, 2019. Accepted for publication April 1, 2020. activator in CD4+ T cells (4, 10), this has mainly been ascribed This work was supported by Versus Arthritis (21139), the King’s Bioscience Institute to its cooperation with other factors, such as FOXP3 (11) and and the Guy’s and St. Thomas’ Charity Prize PhD Program in Biomedical and BLIMP1 in CD4+ regulatory T cells (Tregs) (12), and with STAT3 Translational Science, the Innovative Medicines Institute–funded project Be the Cure (115142-2), and the Department of Health via the National Institute for Health in T follicular helper cells (13). Studies in multiple cell lines Research (NIHR) Comprehensive Biomedical Research Centre award to Guy’s and highlight the ability of IKZF3 to repress gene expression through St. Thomas’ National Health Service (NHS) Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. The HDAC and PRC2 recruitment (14–16) as well as by altering views expressed are those of the author(s) and not necessarily those of the NHS, the chromatin superstructure (17). NIHR, or the Department of Health. Anti–TNF-a mAb therapy is commonly used in the treatment M.L.R. designed and performed experiments, analyzed the data, and wrote the man- of many inflammatory conditions, including rheumatoid arthritis uscript; V.F. and C.A.R. designed and performed experiments and analyzed data; S.L., A.A., A.M.O., K.J.A.S., and G.A.M.P. performed experiments; J.S. and P.L. (18), inflammatory bowel disease (19), and psoriasis (20). Al- provided plasmids and contributed to technical discussions; L.S.T. supervised the though the mechanisms governing its therapeutic effects are still study and contributed to the designing of experiments, data interpretation, and man- not entirely elucidated, multiple effects on the immune system uscript writing. have been reported, including induction of an anti-inflammatory Address correspondence and reprint requests to Dr. Leonie S. Taams, Centre for + Inflammation Biology and Cancer Immunology, Department of Inflammation Biol- CD4 T cell phenotype (21), modulation of innate immune cell ogy, School of Immunology and Microbial Sciences, King’s College London, 1st function (22, 23), and expansion of Tregs (24), in addition to Floor New Hunt’s House, Guy’s Campus, Great Maze Pond, London SE1 1UL, U.K. blocking TNF-a proinflammatory signaling. We previously dem- E-mail address: [email protected] onstrated that patients with rheumatoid arthritis or ankylosing The online version of this article contains supplemental material. spondylitis treated with anti–TNF-a drugs have increased fre- Abbreviations used in this article: ADA, adalimumab; qPCR, quantitative PCR; Treg, quencies of IL-10+ CD4+ T cells in peripheral blood (10). Fur- regulatory T cell. thermore, CD4+ T cells from the peripheral blood of healthy This article is distributed under the terms of the CC BY 4.0 Unported license. volunteers activated in the presence of anti–TNF-a therapeutics had + Copyright Ó 2020 The Authors increased frequencies of IL-10 cells (10, 25). Gene expression www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901283 2 IKZF3 IS ASSOCIATED WITH IL-10+ CD4+ T CELLS analysis from one of these studies highlighted IKZF3 as a potential (PeproTech) for 24 h at a density of 1 3 106 cells/ml. Viral supernatants regulator of IL-10 expression, at least in Th17 cells (10). were mixed with TransDux MAX (Cambridge Bioscience), added to the In this study we sought to address the hypothesis that IKZF3 is a cells, and cultured. After 3 d, the cells were supplemented with fresh 10% + FCS RPMI 1640 and 20 U/ml recombinant human IL-2 and rested from transcriptional regulator of IL-10 production in CD4 T cells. stimulation for 3 d. These cells were subsequently sorted on live CD3+ GFP+/2 cells (Supplemental Fig. 1D). Cells were rested overnight at a 5 Materials and Methods density of 1 3 10 cells/ml, then stained for IL-10, IL-17A, IFN-g, and IKZF3. Cells and cell culture Plasmids and cloning Peripheral blood was obtained from healthy adult volunteers with written informed consent (Bromley Research Ethics Committee ref- The selected regions of the human IL10 locus (indicated in Table II) were erence 06/Q0705/20). PBMCs were isolated using density gradient amplified by PCR using the BAC RP11-262N9 (Thermo Fisher Scientific) + + centrifugation. CD4 T cells and CD14 monocytes were isolated as a template, and TOPO-cloned into pCR-Blunt II-TOPO (Invitrogen). + by MACS using the manufacturer’s protocol. CD14 monocytes These were then sequenced to confirm 100% conformity to the reference + were isolated using anti-CD14 microbeads to ∼98% purity (Miltenyi sequence.
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