The TIGIT/CD226 Axis Regulates Human T Cell Function Ester Lozano, Margarita Dominguez-Villar, Vijay Kuchroo and David A. Hafler This information is current as of September 26, 2021. J Immunol 2012; 188:3869-3875; Prepublished online 16 March 2012; doi: 10.4049/jimmunol.1103627 http://www.jimmunol.org/content/188/8/3869 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2012/03/16/jimmunol.110362 Material 7.DC1 References This article cites 24 articles, 8 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/188/8/3869.full#ref-list-1 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 26, 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 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 The TIGIT/CD226 Axis Regulates Human T Cell Function Ester Lozano,*,†,‡ Margarita Dominguez-Villar,*,†,‡ Vijay Kuchroo,‡,1 and David A. Hafler*,†,‡,1 T cell Ig and ITIM domain (TIGIT) is a newly identified receptor expressed on T cells that binds to CD155 on the dendritic cell surface, driving them to a more tolerogenic phenotype. Given that TIGIT contains an ITIM motif in its intracellular domain and considering the potential importance of the TIGIT/CD226 pathway in human autoimmune disease, we investigated the specific role of TIGIT in human CD4+ T cells. Using an agonistic anti-TIGIT mAb, we demonstrate a direct inhibitory effect on T cell proliferation with a decrease in expression of T-bet, GATA3, IFN regulatory factor 4, and retinoic acid-related orphan receptor c with inhibition of cytokine production, predominantly IFN-g. Knockdown of TIGIT expression by short hairpin RNA resulted in an increase of both T-bet and IFN-g mRNA and protein expression with concomitant decrease in IL-10 expression. Increases in IFN-g with TIGIT knockdown could be overcome by blocking CD226 signaling, indicating that TIGIT exerts immunosuppressive effects by competing with CD226 for the same CD155 ligand. These data demonstrate that TIGIT can inhibit T cell functions by Downloaded from competing with CD226 and can also directly inhibit T cells in a T cell-intrinsic manner. Our results provide evidence for a novel role of this alternative costimulatory pathway in regulating human T cell responses associated with autoimmune disease. The Journal of Immunology, 2012, 188: 3869–3875. cells are driven into cell cycle after TCR recognition of genesis. This includes allelic variants in the CTLA4/CD86 pathway Ag presented by MHC and a second signal first eluci- (3–5) and CD226 that has been associated with risk to develop both http://www.jimmunol.org/ T dated with the CD80/CD86–CD28 costimulatory path- type 1 diabetes and multiple sclerosis (MS) (6, 7). Costimulatory way (1). Although this has become a major tenant in immunology, pathways regulate the functional outcome of T cell activation, and it has become clear that there are a multitude of signals integrated allelic variations altering the balance between positive and negative by the T cell ultimately leading to either entry into cell cycle, costimulatory signals may increase the risk of autoimmune diseases expansion, and differentiation or to nonresponsiveness or apopto- (2). These genetic investigations may be particularly useful in fo- sis. Curiously, many of these costimulatory pathways have mul- cusing on key nodal points that modulate immune response. tiple receptor/ligand pairs, one of which results in positive sig- CD226 was first identified by Shibuya et al. (8, 9) as having naling events while the other transmits negative signals, such as a role in enhancing cytotoxic function of NK cells, demonstrating CD28/CTLA4 (2). A first step in deciphering how T cells integrate that CD226 is associated with LFA-1 to induce IFN-g production by guest on September 26, 2021 these second signals requires an understanding of the individual in naive CD4+ T cells. CD226 binds to two different cell surface components of the costimulatory pathways. ligands, CD155 (poliovirus receptor) and CD112 (10, 11). T cell Genome-wide association scans in patients with autoimmune dis- Ig and ITIM domain (TIGIT) is a transmembrane glycoprotein eases have identified allelic variants in a number of T cell costimula- belonging to a poliovirus receptor family of type 1 proteins that tory molecular pathways as genetic risk factors for disease patho- also binds to CD155 and CD112 ligands (12). TIGIT is expressed on peripheral memory and regulatory CD4+ T cells and NK cells and is upregulated following activation on naive CD4+ T cells. *Department of Neurology, Yale School of Medicine, New Haven, CT 06520; †Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520; CD155 is a high-affinity receptor for TIGIT expressed on mono- + and ‡Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 cytes and CD11c human dendritic cells (DCs) (12). TIGIT con- 1V.K. and D.A.H. contributed equally to this work. tains an Ig-like V-type domain and an ITIM in its cytoplasmic Received for publication December 15, 2011. Accepted for publication February 12, domain, suggesting that receptor occupancy may trigger a nega- 2012. tive signaling event. Similar to the costimulatory CD28/CTLA4– This work was supported by National Institutes of Health Grants U19AI070352, CD80/CD86 pathway where CD28 engagement induces positive R01NS024247, P01AI03971, and P01NS038037 (to D.A.H.). D.A.H. is a Jacob Jav- and CTLA4 negative signaling while the CTLA-4/CD28 pathway its Scholar of the National Institute of Neurological Disorders and Stroke. E.L. was supported by Ministerio de Educacio´n y Ciencia/Fulbright y Ca´tedras “Prı´ncipe de has been intensively investigated, little is known regarding the Asturias” 2008–2011 and is a recipient of a postdoctoral scholarship in the Beatriu TIGIT/CD226 pathway, particularly in human T cells. de Pino´s Programme (2011–2013) (Age`ncia de Gestio´ d’Ajuts Universitaris i de Grogan and colleagues (12) described that engagement of Recerca) with the support of the Commission for Universities and Research of the Ministry of Innovation, Universities and Enterprise of the Autonomous Government TIGIT by CD155 on human DCs enhanced the production of of Catalonia, and the Cofund program of the Marie Curie Actions of the 7th R&D IL-10 while diminishing the production of IL-12p40. In addition Framework Program of the European Union. to effects on APCs, we demonstrated a T cell-intrinsic inhibitory Address correspondence and reprint requests to Dr. David A. Hafler, Department of effect of TIGIT-dependent T cell signaling pathway in murine Neurology, Yale School of Medicine, 15 York Street, PO Box 208018, New Haven CT 06520-8018. E-mail address: david.hafl[email protected] models of experimental autoimmune encephalomyelitis (EAE) The online version of this article contains supplemental material. (13). Specifically, we demonstrated that loss of TIGIT expression Abbreviations used in this article: DC, dendritic cell; IRF, IFN regulatory factor; MS, in mice results in hyperproliferative T cell responses and increased multiple sclerosis; RORc, retinoic acid-related orphan receptor c; sh, short hairpin; susceptibility to EAE. By generating an agonistic anti-TIGIT siCD226, CD226-specific small interfering RNA; siRNA, small interfering RNA; mAb, we showed that TIGIT directly inhibited murine T cell TIGIT, T cell Ig and ITIM domain. responses even in the absence of APCs, demonstrating a T cell Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 intrinsic inhibitory effect of TIGIT. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103627 3870 THE TIGIT/CD226 AXIS REGULATES HUMAN T CELL FUNCTION Considering the potential importance of the TIGIT/CD226 with a Live/Dead Fixable Dead Cell Stain Kit (Molecular Probes) was pathway in human autoimmune disease and based on our mu- performed before fixation to allow gating on viable cells. rine studies (13), we investigated the specific role of TIGIT in ELISA human CD4+ T cell activation in the absence of APCs where CD155 expression on T cells provided a costimulatory signal. ELISA measurement of IFN-g and IL-10 were performed with purified coating and biotinylated detection Abs: human IFN-g mAb (clone 2G1) Using an agonistic anti-TIGIT mAb, we demonstrate a direct in- and human IFN-g mAb biotin-labeled (Thermo Scientific, Rockford, IL) hibitory effect on T cell proliferation with a decrease in expression and rat anti-human and viral IL-10 and biotin anti-human and viral IL-10 of T-bet, GATA3, IFN regulatory factor (IRF)4, and retinoic acid- (BD Biosciences). related orphan receptor c (RORc) with inhibition of cytokine Quantification of mRNA expression levels by real-time PCR production, predominantly IFN-g. Knockdown of TIGIT expres- sion by short hairpin (sh)RNA expression constructs introduced RNAwas isolated using a Qiagen RNeasy Micro kit (Qiagen, Valencia, CA) + and converted to cDNA by reverse transcription with random hexamers and into human CD4 T cells resulted in an increase of both T-bet and MultiScribe reverse transcriptase (TaqMan reverse transcription reagents; IFN-g mRNA and protein expression. These effects could be Applied Biosystems, Foster City, CA).