Downregulation of Inflammatory MicroRNAs by Ig-like Transcript 3 Is Essential for the Differentiation of Human CD8 + T Suppressor Cells This information is current as of September 27, 2021. Chih-Chao Chang, Qing-Yin Zhang, Zhuoru Liu, Raphael A. Clynes, Nicole Suciu-Foca and George Vlad J Immunol 2012; 188:3042-3052; Prepublished online 2 March 2012; doi: 10.4049/jimmunol.1102899 Downloaded from http://www.jimmunol.org/content/188/7/3042 Supplementary http://www.jimmunol.org/content/suppl/2012/03/02/jimmunol.110289 http://www.jimmunol.org/ Material 9.DC1 References This article cites 46 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/188/7/3042.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Downregulation of Inflammatory MicroRNAs by Ig-like Transcript 3 Is Essential for the Differentiation of Human CD8+ T Suppressor Cells Chih-Chao Chang, Qing-Yin Zhang, Zhuoru Liu, Raphael A. Clynes, Nicole Suciu-Foca, and George Vlad We have investigated the mechanism underlying the immunoregulatory function of membrane Ig-like transcript 3 (ILT3) and soluble ILT3Fc. microRNA (miRNA) expression profile identified genes that were downregulated in ILT3-induced human CD8+ T suppressor cells (Ts) while upregulated in T cells primed in the absence of ILT3. We found that miR-21, miR-30b, and miR-155 target the 39-untranslated region of genes whose expression was strongly increased in ILT3Fc-induced Ts, such as dual specificity phosphatase 10, B cell CLL/lymphoma 6, and suppressor of cytokine signaling 1, respectively. Transfection of miRNA mimics or inhibitors and Downloaded from site-specific mutagenesis of their 39-untranslated region binding sites indicated that B cell CLL/lymphoma 6, dual specificity phosphatase 10, and suppressor of cytokine signaling 1 are direct targets of miR-30b, miR-21, and miR-155. Primed CD8+ T cells transfected with miR-21&30b, miR-21&155, or miR-21&30b&155 inhibitors displayed suppressor activity when added to autolo- gous CD3-triggered CD4 T cells. Luciferase reporter assays of miR-21 and miR-155 indicated that their transcription is highly dependent on AP-1. Analysis of activated T cells showed that ILT3Fc inhibited the translocation to the nucleus of the AP-1 subunits, FOSB and c-FOS, and the phosphorylation of ZAP70 and phospholipase C-g 1. In conclusion, ILT3Fc inhibits T cell activation and http://www.jimmunol.org/ induces the generation of Ts targeting multiple inflammatory miRNA pathways. The Journal of Immunology, 2012, 188: 3042–3052. he induction of Ag-specific regulatory T cells (Treg) from erated by repeated allostimulation in MLC, or alternatively by primed lymphocytes is a complex process that limits the exposure to some cytokines such as IL-10, IFN-a,IFN-b (3–5), or T “collateral damage” resulting from protective immunity other agents, such as vitamin D receptor agonists (6, 7). Upregu- and inflammatory responses against self- and non–self-Ags. There lation of ILT3 expression or ILT3 transfection of DC results in is ample evidence that dendritic cells (DC) can prevent and inhibit inhibition of CD40 signaling and of NF-kB activation (3). Tol- T cell-mediated effector responses acquiring tolerogenic property erogenic human DC are characterized by a high expression of by guest on September 27, 2021 and inducing anergy or instructing T cells to become suppressor/ ILT3/ILT4 on their membrane and by their capacity to induce regulatory cells (1). Th anergy and the differentiation of Treg/Ts (3, 4). In contrast, The human Ig-like transcript (ILT)3 and 4, also known as LIRB4/ knockdown (KD) of ILT3 from DC (ILT3 KD-DC) increases their LIR5/CD85k and LIRB2/LIR2/CD85D, belong to a family of innate TLR responsiveness, as reflected in synthesis and secretion of immune receptors that are expressed by DC and monocytes (2). proinflammatory cytokines (IL-1a and b, IL-6, and type I IFN) and These ILT receptors display a long cytoplasmic tail containing migration factors CXCL10 and CXCL11. ILT3KD-DC enhance ITIMs, which mediate inhibition of cell activation by recruiting T cell proliferation and secretion of IFN-g and IL-17 when pulsed tyrosine phosphatase Src homology region 2 domain-containing with CMV or used as allostimulators in MLC (8). phosphatase 1 (2). Upregulation of ILT3 and ILT4 on the mem- These data, in conjunction with the finding that CD8+ T cells brane of DC can be induced in a cytokine-independent manner by from rejection-free heart, kidney, or liver transplant recipients direct interaction with human CD8+ T suppressor cells (Ts), gen- induced the upregulation of ILT3/4 in donor APC, substantiate the importance of these inhibitory receptors for maintenance of im- Division of Immunogenetics and Cellular Immunology, Department of Pathology and munologic quiescence (1). Cell Biology, Columbia University, New York, NY 10032 The extracellular domain of ILT3 retains the T cell inhibitory Received for publication October 6, 2011. Accepted for publication January 29, function even upon deletion of the cytoplasmic, ITIM-containing 2012. tail, because DC transfected with a construct made up of only the This work was supported by research funding from Juvenile Diabetes Research extracellular portion were still capable to elicit the differentiation Foundation (1-2008-550) and the Interuniversitary Organ Transplantation Consor- + tium (Rome, Italy). of CD8 Ts (9). On the basis of this finding, we engineered a Address correspondence and reprint requests to Dr. Nicole Suciu-Foca, Department soluble form of ILT3, which was expressed as an ILT3Fc fusion of Pathology and Cell Biology, Columbia University, 630 West 168 Street, VC 15- protein and tested its immunomodulatory activity. This recombi- 204, New York, NY 10032. E-mail address: [email protected] nant protein inhibited primary and secondary T cell responses in The online version of this article contains supplemental material. MLC and blocked the differentiation of CD8+ cytotoxic T cells Abbreviations used in this article: BCL6, B cell CLL/lymphoma 6; DC, dendritic (CTL). Furthermore, it elicited the in vitro and in vivo differen- cell; DUSP, dual specificity phosphatase; FOS, FBJ murine osteosarcoma; hRluc, + luciferase (Renilla); ILT3, Ig-like transcript 3; KD, knockdown; luc, luciferase (Fire- tiation of CD8 Ts, which produced no cytokines, inhibited T cell fly); miRNA, microRNA; PLC, phospholipase C; SOCS1, suppressor of cytokine reactivity and induced the upregulation of ILT3 on priming APC signaling 1; TGFBR2, TGF-bR 2; Treg, T regulatory cell; TOB1, transducer of (9–11). In vivo studies showed that ILT3Fc induced tolerance to ERBB2; Ts, T suppressor cell; 39-UTR, 39-untranslated region. allogeneic human pancreatic islet cells transplanted in humanized Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 diabetic NOD/SCID mice (10, 11). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102899 The Journal of Immunology 3043 Gene profile analysis of CD8+ T cells primed for 7 d in MLC in CFSE-labeled CD4 Th responders was assessed at day 3 analyzing dye di- the presence or absence of ILT3Fc showed that several hundred lution by flow cytometry using a FACSCalibur instrument and CellQuest genes belonging to .28 gene ontology categories were modulated software (BD Biosciences). As controls, conditions were set up, in which no CD8 T cells, or no anti-CD3 mAb were added. CD8 T cells transfected with by ILT3Fc (12). There was a striking increase in the expression of control RNA (no miRNA inhibitors) were also assayed. transcription factors belonging to a class of zinc finger transcrip- tional repressors including B cell CLL/lymphoma 6 (BCL6), a Gene promoter and 39-UTR constructs known inhibitor of IFN-g (13), IL-17 (14, 15), IL-5 (16, 17), and Sequences corresponding to 21296 to +40 bp from the mRNA start of granzyme B (18) expression. BCL6 was found to be crucial to CD8 BCL6 and to 2748 to +52 from the RNA start of SOCS1 genes were Ts function because KD of BCL6 from unprimed human CD8 cloned from genomic DNA by PCR using high-fidelity Taq and gene- specific primers (Supplemental Table I). These gene promoters, previ- T cells prevented their differentiation into Ts, whereas transfection ously shown to be functional in response to various stimuli in reporter + of BCL6 in primed CD8 T cells resulted in the acquisition of Ts assays (22, 23), were subsequently cloned into HindIII or BglII and NheI function (11). High expression of BCL6 was also found in hu- sites of pGL3 basic Firefly-luciferase construct (Promega). Sequences manized mice tolerating islet allografts (11). Other genes strongly corresponding to 2410 to +40 bp from the mRNA start of miR-21 pro- moter were similarly constructed. This region includes two AP-1 sites, upregulated by ILT3Fc included the dual specificity phosphatase P D AP-1 (proximal) and AP-1 (distal), shown to be fully functional, PMA (DUSP)8, DUSP10, TGF-bR 2 (TGFBR2), transducer of ERBB2 responsive, and critical for expression of miR-21 in 293T cells (24).