The TIGIT/CD226 Axis Regulates Human 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

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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

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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 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 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 ( receptor) and CD112 (10, 11). T cell Genome-wide association scans in patients with autoimmune dis- Ig and ITIM domain (TIGIT) is a transmembrane eases have identified allelic variants in a number of T cell costimula- belonging to a poliovirus receptor family of type 1 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 (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). For mRNA expression assays, overcome by blocking CD226 signaling, indicating that TIGIT probes were purchased from Applied Biosystems and the reactions run on exerts immunosuppressive effects by competing with CD226 for a 7500 Fast real-time PCR system (Applied Biosystems). To evaluate IL-2 the same ligand CD155. Furthermore, we also show that TIGIT gene expression, memory T cells were incubated in the presence of anti- TIGIT mAb or an isotype control for 2 d (probe Hs_00914135_m1 from expression and function are not altered in untreated relapsing- Applied Biosystems). Values are represented as the difference in Ct values remitting MS patients, suggesting that this pathway could poten- normalized to b2-microglobulin for each sample as per the following 2dCt tially be targeted therapeutically to inhibit the hyperactivated formula: Relative RNA expression = (2 ) 3 1000. Downloaded from + status of memory CD4 T cells in patients. These data provide CD226 silencing by small interfering RNA evidence for a novel role of this alternative costimulatory pathway in regulating human T cell responses associated with autoimmune Predesigned small interfering RNA (siRNA) for human CD226 and a negative control siRNA were obtained from Applied Biosystems. Purified disease. CD4+ T cells (1 3 106) were transfected with either negative control siRNA or CD226 siRNA (s20972) by electroporation, using the Nucleo-

Materials and Methods fector device (Amaxa). Transfected cells were incubated at 37˚C for 30 http://www.jimmunol.org/ min, followed by addition of 20 U/ml human IL-2 and transfer of the cells Cell culture reagents and Abs to a 96-well plate coated with anti-CD3/CD28 mAbs. On days 1 and 2 after Peripheral venous blood was obtained from healthy control volunteers in transfection, cells were collected for RNA extraction to assess gene ex- compliance with Institutional Review Board protocols at Yale University pression. On day 3, CD226 surface expression and IFN-g production were School of Medicine. Total CD4+ T cells were isolated by negative selection determined. + (CD4 T cell isolation kit II; Miltenyi Biotec, Auburn, CA) and sorted on Lentiviral shRNA knockdown of CD226 and TIGIT a FACSAria (BD Biosciences, San Jose, CA). Cells were cultured in 96-well plates in RPMI 1640 medium with 5% Lentiviruses expressing shRNAs were obtained from the library of The human AB serum (Gemini Bio-Products, Woodland, CA). The Abs used for RNAi Consortium (15). We initially tested five different hairpins directed stimulation were anti-CD3 (clone UCHT1) and anti-CD28 (clone 28.2) (BD

against CD226 and TIGIT and compared their efficiency by real-time PCR by guest on September 26, 2021 Biosciences) at 1 mg/ml. Recombinant human IL-2 was obtained through against a control hairpin. Tested target sequences are shown in Supple- the AIDS Research and Reference Reagent Program (Division of AIDS, mental Table I; shCD226, shTIGIT-1, and shTIGIT-2 were the most effi- National Institute of Allergy and Infectious Diseases, National Institutes of cient. Health) and was used at 10 U/ml. Human CD4+ T cells were plated on a 96-well plate at a concentration Abs to human TIGIT (VSTM3) were provided by ZymoGenetics (a of 105 cells per well and activated with anti-CD3 and anti-CD28 at 1 mg/ wholly-owned subsidiary of Bristol-Myers Squibb) and were generated by ml. After 24 h, the medium was carefully removed, 22 ml virus and 6 mg/ immunizing BALB/C mice with soluble human TIGIT protein (14). None ml polybrene were added, and the plate was centrifuged at 2250 rpm for 30 of the five mAbs against human TIGIT was capable of blocking the min at room temperature. After centrifugation, all medium was removed binding of soluble human TIGIT to cells expressing human CD155. and fresh supplemented RPMI 1640 medium was added. After 48 h cells However, two of these mAbs showed an agonistic activity measured in were selected with 2 mg/ml puromycin and collected for analysis 72 h after T cell proliferation assays (14). For our functional assays, we use the clone selection. 318.28.2.1 (isotype IgG1) plate-bound at 50 mg/ml. The endotoxin level in the Ab solution was ,1 EU/ml. All Abs were dialyzed before perform- Statistical analysis ing functional assays. Mouse Anti-TIGIT functional grade mAb (clone MBSA43) was obtained from eBioscience (San Diego, CA). Blocking anti- A standard two-tailed Student t test was used for statistical analysis. A p , human CD155 (clone SKII.4) and LEAF purified mouse IgG1 isotype value of 0.05 was considered statistically significant. control were purchased from BioLegend (San Diego, CA), and blocking anti-human CD226 (clone DX11) was from Abcam (Cambridge, MA). Results + Proliferation assay and apoptosis TIGIT, CD155, and CD226 are upregulated on human CD4 T cells after activation CFSE-labeled memory T cells (104/well) were incubated in the presence of plate-bound agonistic anti-TIGIT at 50 mg/ml or IgG isotype control. Cells To investigate the role of TIGIT on T cell function, we began by were stimulated with anti-CD3, anti-CD28, and IL-2 (10 U/ml). Fluores- examining TIGIT expression in human CD4+ T cells ex vivo cence was assessed on day 5 of stimulation on a LSRII flow cytometer (BD and after activation with anti-CD3 and anti-CD28 mAbs. As Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, shown in Fig. 1, TIGIT was not expressed on ex vivo naive T cells OR). Apoptosis was measured with an FITC annexin V Apoptosis detec- 2 tion kit I (BD Biosciences) according to the manufacturer’s instructions. (CD4+CD45RO ), but its expression was induced upon stimula- tion in the CD45RO+ population. Similarly, we confirmed the Cell activation and intracellular staining upregulation of CD226 in both naive and memory populations. Cell populations were stimulated with anti-CD3, anti-CD28, and IL-2 in the Interestingly, CD155, the ligand for both CD226 and TIGIT, was presence of agonistic anti-TIGIT. At day 4, cells were restimulated with also induced after activation both on naive and memory T cells, PMA (50 ng/ml) and ionomycin (250 ng/ml) for 4 h in the presence of although there was a higher frequency of CD155+ T cells in the GolgiStop (BD Biosciences). Cells were fixed and made permeable (Foxp3 2 staining buffer set; eBioscience) and incubated with anti–IFN-g (clone CD45RO population (Fig. 1A). We confirmed upregulation of B27), anti–IL-13 (clone JES10-5A2), anti–IL-4 (clone 8D4-8), anti–IL-9 TIGIT, CD226, and CD155 gene expression by real-time RT-PCR (clone MH9A4), and anti–IL-17 (clone BL168) from BioLegend. Staining on FACS-sorted memory T cells (Fig. 1B). The second ligand for The Journal of Immunology 3871 Downloaded from

FIGURE 1. Upregulation of TIGIT, CD226, and CD155 in human CD4+ T cells after activation. (A) Dot plots displaying the cell surface expression of TIGIT, CD226, CD155, and CD112 on naive CD4+ T cells (CD45RO2) and memory T cells (CD45RO+) ex vivo and after activation with anti-CD3 http://www.jimmunol.org/ and anti-CD28 for 2 d. (B) Kinetics of gene expression of TIGIT, CD226, and their ligands CD155 and CD112 on sorted memory T cells (CD4+ + low + CD62L CD25 CD45RO ). Data are normalized relative to b2-micro- globulin. Data are representative of four donors.

CD226 and TIGIT, CD112, was not expressed on CD4+ T cells ex FIGURE 2. TIGIT cell-intrinsic signaling inhibits T cell proliferation. vivo and was not upregulated upon activation. (A) Proliferation of human CD4+ T cells labeled with CFSE and activated with anti-CD3 and anti-CD28 with plate-bound agonistic anti-TIGIT or

TIGIT signaling inhibits T cell proliferation isotype-matched control Ab. On day 5, CFSE dilution was analyzed by by guest on September 26, 2021 Because TIGIT contains an ITIM that could trigger a negative flow cytometry after gating on viable cells. (B) Percentages of dividing low regulation of T cell functions, we investigated whether TIGIT can cells after activation represent the CFSE cells (n = 8). Mean values are 6 C D also directly inhibit T cell proliferation. T cells were stimulated in shown SD. ( ) Viability was assessed in all of the experiments. ( )No significant differences were observed in the frequency of viable cells. (E) the presence of an agonistic anti-TIGIT mAb (clone 318.28.2.1) for Flow cytometry of the surface expression of CD25 and CD69 as markers of 5 d, and T cell proliferation was assessed by a CFSE dilution assay activation of human CD4+ T cells activated in presence of agonistic anti- after gating on viable cells. As depicted in Fig. 2, this agonistic TIGIT or isotype control, assessed on day 5 after activation. (F) Sorting + anti-TIGIT mAb efficiently inhibited human CD4 T cell prolif- strategy for the purity of memory T cells (CD4+CD62L+CD25low eration (Fig. 2A, 2B). No significant differences were observed in CD45RO+). (G) CFSE proliferation assay of memory T cells in the pres- frequency of viable cells (Fig. 2C, 2D). Recently, a new functional ence of agonistic anti-TIGIT. Proliferation was assessed on day 5 after Ab against human TIGIT (clone MBSA43) has been generated; gating on viable cells. Percentages of proliferating cells are depicted above when we tested this Ab in the same conditions, we did not find an the histograms. (H) Quantitative real-time RT-PCR analysis of relative IL- effect on T cell proliferation, although in certain conditions we 2 gene expression in FACS-sorted memory T cells. Data shown are rep- , observed a slight increase in IFN-g production (Supplemental Fig. resentative of four different donors. *p 0.05. 1). Furthermore, TIGIT ligation did not cause a significant in- crease in the frequency of early or late apoptotic cells (Supple- mental Fig. 2). To address the mechanism by which TIGIT cantly downregulated in the presence of anti-TIGIT (clone engagement leads to inhibition of proliferation, we evaluated the 318.28.2.1) (Fig. 2H), consistent with the decrease in TCR sig- expression of cell surface markers of activation by flow cytometry. naling after TIGIT ligation. Taken together, these data indicate the When primary CD4+ T cells were cultured in the presence of the engagement of TIGIT by its ligands can deliver an inhibitory agonistic anti-TIGIT (clone 318.28.2.1), they showed decreased signal, resulting in decreased T cell activation, IL-2 production, expression of CD25 and CD69 as compared with isotype control and TCR-mediated proliferation. (Fig. 2E). To exclude the possibility that anti-TIGIT could act on Furthermore, no significant differences were found in TIGIT the small percentage of regulatory T cells increasing suppression, expression on ex vivo and activated FACS-sorted memory T cells we isolated CD4+CD62L+CD45RO+CD25low memory T cells by from untreated relapsing-remitting MS patients compared with FACS sorting (Fig. 2F) and repeated proliferation assays exclud- age-matched healthy donors (Supplemental Fig. 3). In agreement ing any other cell type. We confirmed that TIGIT has an intrinsic with the results obtained with healthy donor T cells, the inhibitory inhibitory effect on human memory T cell proliferation (Fig. 2G). effects of TIGIT signaling are functional in T cells from MS To better characterize the mechanism by which TIGIT decreases patients, suggesting that activation of TIGIT signaling may rep- T cell proliferation, we also quantified the production of IL-2 resent a new approach for the treatment of human autoimmune by real-time PCR. Importantly, IL-2 gene expression was signifi- disease. 3872 THE TIGIT/CD226 AXIS REGULATES HUMAN T CELL FUNCTION

FIGURE 4. Knockdown of CD226 expression in human CD4+ T cells. (A) CD226 gene expression in human CD4+ T cells transfected with Downloaded from specific siCD226 or negative siRNA, assessed by real-time RT-PCR on days 1 and 2 after electroporation. (B) Flow cytometry of the surface ex- + FIGURE 3. TIGIT cell-intrinsic signaling inhibits T cell cytokine pro- pression of CD226 and IFN-g intracellular staining on human CD4 duction. (A) Time course of the expression of transcription factors in T cells transfected with siRNA or control, and stimulated with PMA and memory T cells stimulated with IL-12 (Th1), IL-4 (Th2), TGF-b plus IL-4 ionomycin for 4 h in the presence of GolgiStop. Cells were stained with a Live/Dead Fixable Dead Cell Stain Kit before fixation to allow gating on

(Th9), and TGF-b plus IL-1b/IL-6/IL-21 (Th17) and cultured in the http://www.jimmunol.org/ C presence of agonistic anti-TIGIT or isotype control for 3 d. (B) Intracel- viable cells. ( ) Measurement of IFN-g in the supernatants of cells transfected with siCD226 or negative siRNA. (D) Flow cytometry of the lular cytokine staining for IFN-g, IL-13, IL-4, IL-9, and IL-17 in memory + T cells cultured in the same conditions as in (A) for 4 d. Data shown are surface expression of CD226 and IFN-g intracellular staining on CD4 B E representative of at least four independent experiments. T cells transfected as in ( ) and then stimulated with IL-12 for 3 d. ( ) Flow cytometry of the surface expression of CD226 and IFN-g intracel- lular staining on human CD4+ T cells transduced with shCD226 or control TIGIT signaling inhibits cytokine production by lentiviral transduction. Data are representative of three independent 6 To further study the direct mechanism of the inhibitory effect of experiments. Mean values are shown SEM. TIGIT on T cell function, we then investigated the role of TIGIT signaling on cytokine production by CD4+CD62L+CD45RO+ duced CD226 expression with a concomitant decrease in IFN-g by guest on September 26, 2021 CD25low memory T cells. To specifically investigate whether the production. We observed similar results by measuring IFN-g effect of TIGIT signaling is more prominent for a specific func- levels in the supernatants of cells transfected with siCD226 or tional T cell subset, we incubated memory T cells with plate- siRNA negative (Fig. 4C). After confirming CD226 depletion, bound anti-TIGIT (clone 318.28.2.1) or isotype control mAb cells from replicate wells were stimulated with IL-12 for 3 more and stimulated them in Th1, Th2, Th9, and Th17 conditions for days and we observed that the decrease in CD226 expression 3 d. TIGIT ligation efficiently inhibited expression of transcription correlates with a lower percentage of IFN-g–producing cells (Fig. factors T-bet, GATA3, IRF4, and RORc specific for regulating 4D). We validated these results by lentiviral transduction of Th1, Th2, Th9, and Th17 cells, respectively (Fig. 3A). We next shRNA into human CD4+ T cells to specifically downregulate assessed cytokine production by intracellular staining in anti- CD226 expression. We used the vector pLKO-1, containing a gene TIGIT–treated cells. Consistent with the decrease in T-bet ex- encoding puromycin resistance, which allowed us to select T cells pression, the frequency of IFN-g–producing T cells was signifi- with stable integration of shRNA constructs. We tested five dif- cantly reduced with signaling by anti-TIGIT mAb (Fig. 3B). To ferent shRNA constructs to knockdown CD226, using the target a lesser extent, production of IL-13, IL-9, and IL-17 was also sequence indicated as shCD226, the most efficient in depleting reduced in T cells stimulated with anti-TIGIT mAb for 4 d. These CD226 expression (Supplemental Table I). The decrease in data clearly demonstrate that TIGIT suppresses T cell responses CD226 cell surface expression resulted in an ∼27% reduction in directly in a T cell-intrinsic manner. IFN-g–producing cells (Fig. 4E). Taken together, our data strongly suggest that CD226, possibly through its interaction with CD155, CD226 knockdown decreases T-bet expression and IFN-g can trigger a positive signal in T cells inducing T-bet expression production in human CD4+ T cells and IFN-g production. In addition to the inhibitory cell-intrinsic role for TIGIT on T cells, TIGIT knockdown increases T-bet expression and IFN-g we wanted to examine whether TIGIT has a second mechanism + of negative immunoregulation by competing with CD226 for the production in human CD4 T cells same CD155 ligand. To specifically explore the contribution of To better understand the role of TIGIT in this regulatory pathway, the CD226 and TIGIT receptors, we performed an siRNA knock- we used a specific shRNA to stably downregulate TIGIT expres- down of CD226 on ex vivo CD4+ T cells. A decrease in CD226 sion in human CD4+ T cells. We tested five different shRNA con- expression resulted in a decrease in T-bet and IFN-g mRNA ex- structs to knockdown TIGIT; one directed against the 39 untrans- pression (Fig. 4A). On day 3, we stimulated cells with PMA and lated region of TIGIT mRNA (shTIGIT-1) and the other against ionomycin for 4 h and analyzed CD226 surface expression versus the coding sequence (shTIGIT-2) that were efficient in downreg- IFN-g production, gating on viable cells. As shown in Fig. 4B, ulating TIGIT expression (Supplemental Table I). On day 3 after CD226-specific small interfering RNA (siCD226) effectively re- puromycin selection, shTIGIT-1 and shTIGIT-2 decreased the The Journal of Immunology 3873

+ FIGURE 5. Knockdown of TIGIT expression in human CD4 T cells by lentiviral transduction with two specific shRNAs. (A) Quantitative real-time RT- Downloaded from PCR analysis of relative TIGIT gene expression in human CD4+ T cells infected with lentivirus containing two TIGIT-specific shRNA sequences or an empty vector, assessed on day 3 after puromycin selection. (B) Gene expression of other negative regulators, CTLA4 and PD-1, was measured to exclude nontarget effects. (C) Relative quantification of T-bet (Tbx21), IFN-g, and IL-10 mRNA in T cells transduced with shTIGIT-1, shTIGIT-2, or an empty vector. (D) Flow cytometry of the surface expression of TIGIT and IFN-g intracellular staining on human CD4+ T cells infected with TIGIT-specific shRNA or control, selected with puromycin and stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. Cells were stained with a Live/Dead Fixable Dead Cell Stain Kit before fixation to allow gating on viable cells. Data are representative of four independent experiments with three individual donors. (E) Levels of IL-10 in the supernatants derived from the same cultures as in (C) quantified by ELISA. Mean values are shown 6 SEM. http://www.jimmunol.org/ expression of TIGIT mRNA .80% (Fig. 5A) but not of other CD226/CD155 provides positive costimulation to induce IFN-g negative T cell regulators such as CTLA4 and PD-1 (Fig. 5B). production whereas TIGIT is required for IL-10 production. To determine the effect of knocking down TIGIT expression on cytokine production, we examined the expression of transcription Discussion factor T-bet (Tbx21), the master transcription factor that induces Allelic variants in the TIGIT/CD226 pathway have recently been IFN-g production. Knockdown of TIGIT mRNA expression was identified as risk factors for developing human autoimmune dis- accompanied by increases in both T-bet and IFN-g mRNA ex- by guest on September 26, 2021 pression (Fig. 5C). In contrast, deletion of TIGIT resulted in a significant reduction of IL-10 gene expression in cells trans- duced with both shTIGIT-1 and shTIGIT-2 (Fig. 5C), suggesting a central role of TIGIT in reciprocally regulating proinflammatory (IFN-g) and anti-inflammatory (IL-10) cytokines. To confirm these results at the protein level, we stimulated the puromycin-resistant cells with PMA and ionomycin for 4 h and then assayed TIGIT expression and cytokine production by in- tracellular staining. Consistent with a role of TIGIT in reducing T cell responses, knockdown of TIGIT increased IFN-g production in cells transduced with shTIGIT-1 and shTIGIT-2 (Fig. 5D), confirming that TIGIT may act directly by inhibiting IFN-g pro- duction in human T cells. Measurement of IL-10 secretion in transduced CD4+ T cells revealed that depletion of TIGIT was accompanied by a decrease in IL-10 production (Fig. 5E), sug- gesting a cell-intrinsic role for TIGIT in IL-10 production in the absence of APCs. IFN-g secretion after TIGIT knockdown is mediated by CD155 engagement of CD226 To examine the mechanism for the increased production of IFN-g with knockdown of TIGIT, we cultured with mAbs directed against either CD155 or CD226 that were expressed on T cells. As shown in Fig. 6, adding either anti-CD155 or anti-CD226 mAbs blocked FIGURE 6. Blockade of CD226 signaling with anti-CD226 or anti-CD155 the increased T-bet and IFN-g gene expression induced by TIGIT decreased the effects of TIGIT knockdown. (A) Quantitative real-time PCR knockdown. Interestingly, blockade of CD226 or CD155 also de- analysis of relative T-bet (Tbx21) expression in TIGIT-depleted CD4+ Tcells creased IL-10 gene expression (Fig. 6C). These data indicate that in incubated for 4 d in the presence of increasing concentrations of plate-bound the absence of TIGIT, CD226/CD155 interaction induces T-bet blocking anti-CD226, blocking anti-CD155, or isotype control IgG. (B) and IFN-g production, whereas IL-10 production requires TIGIT IFN-g gene expression. (C) IL-10 gene expression in the same conditions for its induction. These data further support our hypothesis that as in (A). Data shown are representative of four different donors. 3874 THE TIGIT/CD226 AXIS REGULATES HUMAN T CELL FUNCTION eases (7). However, the mechanisms associated with human CD4+ of an Ab against CTLA4 (ipilimumab) that enhances immune acti- T cell expression of these receptors are not known. In this study, vation against cancer cells, resulting in significant antitumor ef- we demonstrate that engagement of TIGIT with an agonistic mAb fects (22). triggers a direct inhibitory effect on T cell entry into cell cycle In this study, we demonstrated that TIGIT signaling could also with a decrease in expression of the T-bet, GATA3, IRF4, and directly inhibit T cell activation by a second mechanism where RORc transcription factors associated with inhibition of cytokine TIGIT acts as negative regulator by competing with CD226 for production, predominantly IFN-g. TIGIT expression represses T- binding to its ligand CD155 to inhibit T cell responses. This new bet, as knockdown of TIGIT induces T-bet–mediated IFN-g se- costimulatory pathway is similar to the CD28/CTLA4 pathway that cretion while decreasing IL-10. The increase in IFN-g was over- binds to B7-1 (CD80) and B7-2 (CD86) ligands. Interestingly, come by blocking CD226 signaling, indicating that TIGIT exerts CTLA4 interacts with at least 20-fold higher avidity than CD28 immunosuppressive effects by competing with CD226 for the does (23). In a similar manner, TIGIT is a negative regulator that CD155 ligand similar to the CD28/CTLA4 pathway. These data binds CD155 with higher affinity (119 nM) compared with the provide evidence for a novel role of this alternative costimulatory CD226/CD155 interaction (1–3 nM) (12). Therefore, once ex- pathway in differentially regulating proinflammatory (IFN-g) pressed, TIGIT can effectively compete for binding to the same versus anti-inflammatory (IL-10) cytokine production in a T cell- CD155 ligand similar to CD28/CTLA4 interactions with B7-1/B7- intrinsic manner. 2. In this regard, therapies based on interfering with the costim- Our recent investigations in the EAE model of multiple sclerosis ulatory CTLA4 pathway have recently been approved for clinical have demonstrated a critical role of TIGIT as a negative regulator use. Specifically, a CTLA4 fusion protein (abatacept) has been of autoimmune responses (13). The phenotype of TIGIT-deficient approved to treat rheumatoid arthritis (24). Thus, the TIGIT/ Downloaded from mice includes augmented T cell proliferation upon immuniza- CD226 pathway is an attractive target for therapeutic manipula- tion and higher production of proinflammatory cytokines leading tion. to greater EAE susceptibility (13). These data in the EAE model In addition to its immunosuppressive effects on DCs, in this were confirmed in a collagen-induced arthritis model of arthritis study we demonstrate that TIGIT exerts multiple mechanisms of where administration of soluble TIGIT Fc-fusion protein signifi- peripheral tolerance: 1) the direct inhibition of T cell proliferation

cantly inhibited the severity of disease whereas a blocking anti- and production of proinflammatory cytokines; 2) induction of anti- http://www.jimmunol.org/ TIGIT accelerates disease development (14). Taken together, these inflammatory cytokines such as IL-10; and 3) the blockade of studies show an intrinsic T cell function of TIGIT in regulating CD226-positive costimulatory signaling. To better understand the autoimmune responses and indicate a key role for the TIGIT/ role of each receptor in this complex network, we performed these CD155 pathway in autoimmune diseases. experiments in the absence of APCs. Given that T cells can express In humans, it has been reported that TIGIT expressed on T cells CD155 but not CD112, we can exclude that the observed effects only exerts its immunosuppressive effects indirectly by modulating are due to CD112 signaling. To dissect the underlying molecular cytokine production by DCs (12). In that study, Yu et al. (12) mechanisms of this regulatory pathway, we first downregulated concluded that TIGIT does not have a T cell-intrinsic function. CD226 expression on human T cells and observed a decrease in T- The likely explanation for the discrepancy between their results bet expression and IFN-g production. This result is consistent with by guest on September 26, 2021 and the present study is that the anti-TIGIT mAb (10A7) was not previous reports showing that introduction of mutant (Y-F322) agonistic and perhaps recognized a different TIGIT determinant. CD226 into naive CD4+ T cells suppresses their growth initiated In another study performed by Levin et al. (14), two of five anti- by CD3 and LFA-1 ligations in the absence of IL-2, suggesting human TIGIT showed an agonistic effect efficiently inhibiting that CD226 functions as a signal transducer of LFA-1 upon trig- T cell proliferation. In our investigations, one of these clones was gering T cell activation (9). When we specifically knocked down selected for functional assays (clone 318.28.2.1) whereas the TIGIT expression, we obtained the opposite results: an increase in others could bind TIGIT but did not show any agonistic effect. T-bet and IFN-g expression, consistent with the inhibitory effects Consistent with data reported by Levin et al. (14), we demon- triggered by the agonistic anti-TIGIT mAb. These data demon- strated that engagement of TIGIT by its ligands can deliver an strate that TIGIT ligation initiates a negative signaling cascade inhibitory signal resulting in decreased T cell activation, IL-2 and by blocking CD226 or CD155 in TIGIT-depleted cells, and production, and TCR-mediated proliferation. Thus, the agonistic the increases in IFN-g production are due to an enhanced CD226/ function of the TIGIT mAbs in vitro and in vivo in the EAE model CD155 interaction. suggests its utility therapeutically, particularly with our recent Interestingly, lack of TIGIT resulted in a decrease in IL-10 finding of Th1 regulatory T cells in patients with MS where IFN-g production. Accordingly, studies in TIGIT2/2 mice showed that expression inhibits the function of regulatory T cells (16). IL-10 was not induced by Ag-specific stimulation in TIGIT2/2 The TIGIT-intrinsic mechanism of negative regulation of T cell cultures, suggesting that production of IL-10 by T cells was T cell functions may also have implications in tumor immunology. impaired (13). One possible explanation for this defect in IL-10 Similar to PD-1 ligands, TIGIT ligands CD155 and CD112 are production could be that TIGIT signaling directly activates IL- overexpressed in certain human tumors, including colorectal car- 10 production but agonistic anti-TIGIT mAb failed to increase cinomas (17), gastric cancer (18), and neuroblastomas (19). IL-10 expression (data not shown). Alternatively, in the absence of CD226/CD155 interaction has been implicated as a major trigger TIGIT, CD226 can interact with CD155, increasing IFN-g while for NK cell antitumor activity (20), whereas TIGIT/CD155 in- repressing IL-10 gene expression. However, blocking CD226/ teraction inhibits NK cell cytotoxicity (21). However, the role CD155 interaction did not increase IL-10 expression. It has of TIGIT in tumor settings remains to be elucidated. Our data been reported that TIGIT, through its interaction with CD155, can demonstrate that TIGIT is critical for the regulation of lympho- modulate Erk activity and increase IL-10 production by DCs (12). cyte function and can efficiently limit T cell-dependent immune It is intriguing to speculate that T cells expressing CD155 might responses. This mechanism may enable tumors overexpressing be regulated in a similar manner and thus TIGIT depletion im- CD155 to escape from immune attack. The clinical relevance of paired IL-10 production by CD155-expressing T cells. modulation of a negative regulator to augment immune responses TIGIT/CD226 pathway is a novel regulatory pathway of T cell directed at tumors has been recently proved with the generation function associated with human autoimmune disease. Manipula- The Journal of Immunology 3875 tion of the TIGIT/CD226 axis in vitro regulated T cell function by 10. Bottino, C., R. Castriconi, D. Pende, P. Rivera, M. Nanni, B. Carnemolla, C. Cantoni, J. Grassi, S. Marcenaro, N. Reymond, et al. 2003. Identification of cell-intrinsic and cell-extrinsic mechanisms. To examine the po- PVR (CD155) and -2 (CD112) as cell surface ligands for the human tential therapeutic intervention of this pathway in patients with DNAM-1 (CD226) activating molecule. J. Exp. Med. 198: 557–567. autoimmune diseases, we demonstrate that TIGIT expression is not 11. Tahara-Hanaoka, S., K. Shibuya, Y. Onoda, H. Zhang, S. Yamazaki, A. Miyamoto, S. Honda, L. L. Lanier, and A. Shibuya. 2004. Functional char- altered in untreated relapsing-remitting MS patients and that an acterization of DNAM-1 (CD226) interaction with its ligands PVR (CD155) and + agonistic Ab to TIGIT has an inhibitory effect on CD4 T cells nectin-2 (PRR-2/CD112). Int. Immunol. 16: 533–538. from MS patients. Although further studies are needed to test the 12. Yu, X., K. Harden, L. C. Gonzalez, M. Francesco, E. Chiang, B. Irving, I. Tom, S. Ivelja, C. J. Refino, H. Clark, et al. 2009. The surface protein TIGIT sup- therapeutic benefits of targeting inhibitory pathways, modulation presses T cell activation by promoting the generation of mature immunoregu- of the TIGIT/CD226 axis may allow more targeted manipulation latory dendritic cells. Nat. Immunol. 10: 48–57. of human CD4+ T cells in autoimmune diseases. 13. Joller, N., J. P. Hafler, B. Brynedal, N. Kassam, S. Spoerl, S. D. Levin, A. H. Sharpe, and V. K. Kuchroo. 2011. Cutting edge: TIGIT has T cell-intrinsic inhibitory functions. J. Immunol. 186: 1338–1342. Acknowledgments 14. Levin, S. D., D. W. Taft, C. S. Brandt, C. Bucher, E. D. Howard, E. M. Chadwick, J. Johnston, A. Hammond, K. Bontadelli, D. Ardourel, et al. We thank the blood donors and MS patients for participation. Abs to human 2011. Vstm3 is a member of the CD28 family and an important modulator of T- TIGIT were provided by ZymoGenetics/Bristol-Myers Squibb under a Ma- cell function. Eur. J. Immunol. 41: 902–915. terial Transfer Agreement. 15. 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