Regulatory T Cell Expression of Herpesvirus Entry Mediator Suppresses the Function of B and T Lymphocyte Attenuator-Positive Effector T Cells This information is current as of October 1, 2021. Ran Tao, Liqing Wang, Kenneth M. Murphy, Christopher C. Fraser and Wayne W. Hancock J Immunol 2008; 180:6649-6655; ; doi: 10.4049/jimmunol.180.10.6649 http://www.jimmunol.org/content/180/10/6649 Downloaded from References This article cites 34 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/180/10/6649.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 October 1, 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Regulatory T Cell Expression of Herpesvirus Entry Mediator Suppresses the Function of B and T Lymphocyte Attenuator-Positive Effector T Cells1 Ran Tao,* Liqing Wang,* Kenneth M. Murphy,† Christopher C. Fraser,‡ and Wayne W. Hancock2* The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA؊/؊ CD4؉CD25؉ Tregs had normal suppressive activity, whereas BTLA؊/؊ Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, Downloaded from but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM؊/؊ Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM؊/؊ Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo. The http://www.jimmunol.org/ Journal of Immunology, 2008, 180: 6649–6655. he molecular interactions responsible for the suppressive success has been attained using Tregs to achieve long-term allo- activity of CD4ϩCD25ϩ regulatory T cells (Tregs)3 graft survival in normal, immunocompetent hosts (1–3). Therefore, T remain unclear, but Tregs are known to control immune a better understanding of how Tregs work is essential if progress responses to self-Ags, thereby preventing autoimmune disease, toward clinical therapy is to be attained. The detailed mechanisms and to regulate responses to nonself molecules in adaptive im- by which Tregs control immune responses in vivo are unknown. In munity (1). Tregs have received much attention by transplant vitro studies often show a requirement for Treg/T effector (Teff) by guest on October 1, 2021 investigators because allograft tolerance is not routinely cell contact, although functionally active surface molecules on achieved clinically and transplant recipients require lifelong im- Tregs such as CTLA4 (CD152), glucocorticoid-induced TNFR munosuppression and endure varying degrees of associated drug family-related receptor (GITR, TNFRSF18), and the programmed toxicities. The identification and characterization of Tregs led to death receptor PD-1 (CD279), as well as soluble mediators includ- the expectation that these cells would be useful for therapy in ing IL-10 and TGF-␤ can also contribute to Treg suppressive func- transplantation. However, although clinical and experimental stud- tions, depending upon assay conditions and likely other factors (1). ies indicate that manipulating the balance between Tregs and re- Optimal T cell activation requires the TCR engagement of a sponder T cells can dampen host responses posttransplant, little cognate MHC-peptide complex and a costimulatory signal. The binding of CD28 to B7-1/B7-2 remains the best-characterized co- *Department of Pathology and Laboratory Medicine, Joseph Stokes Jr. Research In- stimulatory pathway, but the persistence of T cell responses in Ϫ Ϫ stitute and Biesecker Pediatric Liver Center, The Children’s Hospital of Philadelphia CD28 / mice led to the discovery of several CD28/B7 homologs, † and University of Pennsylvania, Philadelphia, PA 19104; Department of Pathology including the components of the ICOS/B7RP-1 and PD-1/PD-L1/ and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110; and ‡Inflammation, Millennium Pharmaceuticals, PD-L2 pathways (4). The most recently recognized CD28 ho- Cambridge, MA 02139 molog, B and T lymphocyte attenuator (BTLA, CD272) (5), is Received for publication March 9, 2008. Accepted for publication March 9, 2008. unusual in that it interacts with the TNFR superfamily member, The costs of publication of this article were defrayed in part by the payment of page herpesvirus entry mediator (HVEM, TNFRSF14) (6). HVEM can charges. This article must therefore be hereby marked advertisement in accordance promote T cell activation by propagating positive signals from the with 18 U.S.C. Section 1734 solely to indicate this fact. TNF superfamily member ligand, LIGHT (TNFSF14, CD258), a 1 This work was supported in part by Grant R01-AI54720 from the National Institutes of Health (to W.W.H.). lymphotoxin-related inducible ligand that competes for glycopro- 2 Address correspondence and reprint requests to Dr. Wayne W. Hancock, Depart- tein D binding to HVEM on T cells (7). ment of Pathology and Laboratory Medicine, 916B Abramson Research Center, The A 2.8-Å crystal structure of the BTLA-HVEM complex shows Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318. E-mail address: [email protected] that BTLA binds the N-terminal cysteine-rich domain of HVEM and uses a unique binding surface compared with other CD28-like 3 Abbreviations used in this paper: Treg, regulatory T cell; Teff, effector T; GITR, glucocorticoid-induced TNFR family-related receptor; BTLA, B and T lymphocyte receptors (8). The BTLA binding site on HVEM overlaps with the attenuator; HVEM, herpesvirus entry mediator; IRES, internal ribosomal entry site; binding site for the HSV type 1 envelope glycoprotein D, but is WT, wild type. distinct from the binding site for LIGHT. HVEM has three cys- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 teine-rich domains. Competitive binding analysis and mutagenesis www.jimmunol.org 6650 SURFACE MOLECULES MEDIATING Treg SUPPRESSION reveals a unique BTLA binding site centered on a critical lysine and an internal ribosomal entry site (IRES) cassette downstream of the residue in cysteine-rich domain-1, the most membrane-distal do- cloning region and upstream of a cell surface-expressed nonsignaling nerve main, as opposed to LIGHT, which interacts largely through cys- growth factor receptor (15). Plasmid mHVEM-FL-GFP-RV was made from two PCR products, with primers 5Ј-BglII mHVEM and mHVEM/GFP teine-rich domain-3 (8–10). using mHVEM-FL-IRES-GFP-RV as template, and primers mHVEM-GFP The importance of the HVEM-BTLA pathway in control of Teff and 3Ј-GFP plus Sal using mHVEM-FL-IRES-GFP-RV as template; PCR cell functions was shown in several animal models. For example, products were annealed, amplified with primers 5Ј-BglII mHVEM and 3Ј- compared with controls, HVEMϪ/Ϫ mice had increased suscepti- GFP plus Sal, digested with BglII and SalI, and ligated into IRES-GFP-RV that was digested with BglII and SalI. The Phoenix ecotropic packaging bility to Con A mitogen-induced, T cell-dependent autoimmune Ϫ/Ϫ cell line was used for cotransfection with MinR1 Foxp3 and mHVEM hepatitis (11), and BTLA mice had greater acute allergic air- retroviral constructs in the presence of Lipofectamine 2000. Retroviral way inflammation (12), worse myelin oligodendrocyte glycopro- supernatants were added to CD4ϩCD25Ϫ T cells activated overnight tein peptide-induced experimental autoimmune encephalitis (4), with PMA, ionomycin, and IL-2; transduction efficiency was 90–95% and were unable to accept partially MHC-mismatched cardiac al- as determined by flow cytometry, and cells were centrifuged over Ficoll before use. lografts (13). In the current study, we propose a new model in which Tregs exert their suppressive effect via up-regulation of the Cardiac transplantation negative costimulatory ligand HVEM that, upon ligation with Intraabdominal vascularized mouse cardiac transplantation across a full BTLA expressed on the Teff cell side, enhances the suppressive MHC-mismatch was performed using BALB donors and C57BL/6 recip- function of Tregs and helps control immune response in vitro and ients, with anastomoses of donor ascending aorta and pulmonary artery in vivo. end-to-side to recipient infrarenal aorta and inferior vena cava, respectively (13). Grafts were assessed daily by abdominal palpation; rejection was Materials and Methods defined as cessation of cardiac contraction and confirmed by histology.