BTNL2, a Butyrophilin/B7-Like Molecule, Is a Negative Costimulatory Molecule Modulated in Intestinal Inflammation This information is current as Heather A. Arnett, Sabine S. Escobar, Eva Gonzalez-Suarez, of September 28, 2021. Alison L. Budelsky, Lori A. Steffen, Norman Boiani, Ming Zhang, Gerald Siu, Avery W. Brewer and Joanne L. Viney J Immunol 2007; 178:1523-1533; ; doi: 10.4049/jimmunol.178.3.1523 http://www.jimmunol.org/content/178/3/1523 Downloaded from References This article cites 40 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/178/3/1523.full#ref-list-1 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 • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology BTNL2, a Butyrophilin/B7-Like Molecule, Is a Negative Costimulatory Molecule Modulated in Intestinal Inflammation Heather A. Arnett,1* Sabine S. Escobar,1* Eva Gonzalez-Suarez,* Alison L. Budelsky,* Lori A. Steffen,* Norman Boiani,† Ming Zhang,§ Gerald Siu,§ Avery W. Brewer,‡ and Joanne L. Viney2* Butyrophilin-like 2 (BTNL2) is a butyrophilin family member with homology to the B7 costimulatory molecules, polymorphisms of which have been recently associated through genetic analyses to sporadic inclusion body myositis and sarcoidosis. We have characterized the full structure, expression, and function of BTNL2. Structural analysis of BTNL2 shows a molecule with an extracellular region containing two sets of two Ig domains, a transmembrane region, and a previously unreported cytoplasmic tail. Unlike most other butyrophilin members, BTNL2 lacks the prototypical B30.2 ring domain. TaqMan and Northern blot analysis indicate BTNL2 is predominantly expressed in digestive tract tissues, in particular small intestine and Peyer’s patches. Immunohisto- Downloaded from chemistry with BTNL2-specific Abs further localizes BTNL2 to epithelial and dendritic cells within these tissues. Despite its homology to the B7 family, BTNL2 does not bind any of the known B7 family receptors such as CD28, CTLA-4, PD-1, ICOS, or B and T lymphocyte attenuator. Because of its localization in the gut and potential role in the immune system, BTNL2 expression was analyzed in a mouse model of inflammatory bowel disease. BTNL2 is overexpressed during both the asymptomatic and symptomatic phase of the Mdr1a knockout model of spontaneous colitis. In functional assays, soluble BTNL2-Fc protein inhibits the proliferation of murine CD4؉ http://www.jimmunol.org/ T cells from the spleen, mesenteric lymph node, and Peyer’s patch. In addition, BTNL2-Fc reduces proliferation and cytokine produc- tion from T cells activated by anti-CD3 and B7-related protein 1. These data suggest a role for BTNL2 as a negative costimulatory molecule with implications for inflammatory disease. The Journal of Immunology, 2007, 178: 1523–1533. he regulation of T cell responses is achieved through Ag- (4–8) is expressed in both lymphoid and nonlymphoid cells (9). A specific TCRs acting together with coreceptors that can second inhibitory receptor PD-1 (10, 11) binds to either of two T modulate activation. One of the principal costimulatory B7-related ligands PD-L1 (also known as B7-H1) (12, 13) and pathways for naive T cells is initiated when CTLA-4 and CD28 PD-L2 (or B7-DC) (14, 15). Another inhibitory receptor, B and T engage B7 class molecules during TCR engagement by a primary lymphocyte attenuator (BTLA), binds to HVEM, previously iden- by guest on September 28, 2021 signal (1). These secondary costimulatory signals can either inhibit tified as a binding partner for LIGHT and lymphotoxin ␣. B7-H3, or promote T cell responses, depending on the receptor they en- another B7 homolog (16, 17), binds an unidentified receptor on gage. For example, B7-1 and B7-2 can bind either CD28, resulting activated T cells and serves as a negative regulator of T cell acti- in T cell stimulation, or CTLA-4, resulting in T cell inhibition (2). vation (18). A more recently described B7 family member, B7-H4 Negative signals can contribute to the attenuation of T cell re- (also called B7S1 or B7x) (19–21), acts as a novel costimulator sponses and so are important in regulating T cell tolerance. The and regulates the threshold of T cell activation. balance between stimulatory and inhibitory signals is critical to an Butyrophilin-like 2 (BTNL2) has been identified as a B7-like effective immune response. molecule within the butyrophilin gene family, located in the MHC In the past several years, new members of the B7 ligand and the locus in mice and humans (22). Recently, several studies have corresponding CD28/CTLA-4 receptor families have been identi- linked the gene for BTNL2 to two inflammatory diseases, sporadic fied, increasing our understanding of immune cell function. A sec- inclusion body myositis and sarcoidosis (23–25); however, no ond activating receptor in T cells is ICOS (3); its ligand B7-related function has been demonstrated for BTNL2. Although the buty- protein 1 (B7RP1)3 (also known as LICOS, GL50, B7-H2, or B7h) rophilin family of proteins share homology with the B7 family, no immune responses have been associated with BTNL1, the first and most broadly expressed butyrophilin. Because of its close struc- *Inflammation, †Protein Sciences, and ‡Pathology, Amgen, Seattle, WA 98119; and §Inflammation, Amgen, Thousand Oaks, CA 91320 tural homology to B7-1, BTNL2 has been hypothesized to have T cell costimulatory properties (25). Received for publication December 7, 2005. Accepted for publication October 17, 2006. In this study we characterize the expression of BTNL2 in normal The costs of publication of this article were defrayed in part by the payment of page and diseased tissue, and show evidence that BTNL2 down-regulates charges. This article must therefore be hereby marked advertisement in accordance T cell activation and may play a role in maintaining tolerance and with 18 U.S.C. Section 1734 solely to indicate this fact. resetting the balance of the immune response in the gut. 1 H.A.A. and S.S.E. contributed equally to this work. 2 Address correspondence and reprint requests to Dr. Joanne L. Viney, Department of Inflammation, Amgen, 1201 Amgen Court West, Seattle, WA 98119. E-mail address: Materials and Methods [email protected] Cloning 3 Abbreviations used in this paper: B7RP1, B7-related protein 1; BTLA, B and T lymphocyte attenuator; BTNL, butyrophilin-like; MLN, mesenteric lymph node; PP, Murine and human BTNL2 (accession nos. NM_023145 and NM_019602, Peyer’s patch; CHO, Chinese hamster ovary. respectively) were originally identified from the National Center for Bio- technology Information (NCBI) database based on homology to other B7 Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 family molecules. RACE PCR was performed to confirm 5Ј and 3Ј ends of www.jimmunol.org 1524 BTNL2 DOWN-REGULATES T CELL ACTIVATION the human and mouse BTNL2 genes based on the original deposits using 1% Triton X-100, with a protease inhibitor mixture (Boehringer Mann- RACE-Ready Marathon cDNA from human colon and mouse spleen (BD heim). Proteins were separated on 4–20% reducing Tris-glycine gel, trans- Clontech). In addition, cDNAs from human T84 (colon carcinoma) cells, ferred to nitrocellulose, and blocked overnight at 4°C in TBST plus 3% Caco2 (colon carcinoma) cells, and mouse colon were also used to confirm nonfat milk. After washing in TBST, the membranes were incubated with the final cDNA sequences. The final full-length coding region cDNAs of the murine BTNL2–77p polyclonal antisera or control sera for1hat4°C, human and mouse BTNL2 were then amplified as single cDNA contigs, followed by goat anti-rabbit HRP (1/5000; Amersham Biosciences) and with the new additional 3Ј sequence, and then subcloned into pCR4-Topo developed by chemiluminescence (Amersham Biosciences). (Invitrogen Life Technologies) and appropriate mammalian expression vectors. Independent PCR analyses were performed to confirm each final Immunohistochemistry murine and human BTNL2 sequence. The sequence results were compared with both the predicted sequence published by Stammers et al. (22) and the Frozen sections were fixed in acetone at 4°C for 10 min, washed in TBS NCBI database. Transmembrane and signal peptide predictions were predicted (pH 8.0; Sigma-Aldrich) three times, then blocked for endogenous perox- ϩ with TMHMM and SignalP (www.cbs.dtu.dk/services/TMHMM-2.0 and idase in glucose oxidase solution (b-D glucose (G-5250; Sigma-Aldrich), www.cbs.dtu.dk/services/SignalP). glucose oxidase (Sigma-Aldrich), sodium azide (Sigma-Aldrich), and TBS (pH 8.0)) for 30 min at 37°C. Sections were then treated with avidin and Quantitative real-time PCR biotin blocking solutions (Vector Laboratories) for 15 min each, rinsed in TBST and blocked in Tris/NaCl/blocking solution (TNB; PerkinElmer) Expression of murine BTNL2 was measured by quantitative real-time RT- for 30 min. Primary Ab was applied using anti-BTNL2 at 19.5 ␮g/ PCR using the ABI PRISM 7900HT sequence detection system (Applied Bio- ml (xmuRIBD006, rat IgG2a, clone M517; Amgen) or 20 ␮g/ml systems) and normalized to the expression of a housekeeping gene (HPRT).