Cutting Edge: TIGIT Has T Cell-Intrinsic Inhibitory Functions Nicole Joller, Jason P. Hafler, Boel Brynedal, Nasim Kassam, Silvia Spoerl, Steven D. Levin, Arlene H. Sharpe This information is current as and Vijay K. Kuchroo of October 1, 2021. J Immunol 2011; 186:1338-1342; Prepublished online 3 January 2011; doi: 10.4049/jimmunol.1003081 http://www.jimmunol.org/content/186/3/1338 Downloaded from

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: TIGIT Has T Cell-Intrinsic Inhibitory Functions † ‡,x Nicole Joller,* Jason{ P. Hafler, Boel Brynedal,‖ Nasim Kassam,* Silvia Spoerl,* Steven D. Levin, ,1 Arlene H. Sharpe, and Vijay K. Kuchroo* Costimulatory molecules regulate the functional out- through the ligand CD155. TIGIT interaction with CD155 come of T cell activation, and disturbance of the bal- induced tolerogenic DCs that impaired T cell proliferation ance between activating and inhibitory signals results and inhibited IFN-g production from responding T cells (3). in increased susceptibility to infection or the induction However, although TIGIT contains two ITIMs in its cyto- of autoimmunity. Similar to the well-characterized plasmic tail, it has not been elucidated whether TIGIT can CD28/CTLA-4 costimulatory pathway, a newly emerg- directly inhibit T cell responses. This is partly because of the lack of relevant reagents that allow direct evaluation of its role ing pathway consisting of CD226 and T cell Ig and Downloaded from in T cells. ITIM domain (TIGIT) has been associated with sus- 2/2 ceptibility to multiple autoimmune diseases. In this In this study, we used TIGIT mice and generated anti- study, we examined the role of the putative coinhibi- TIGIT mAbs to analyze the function of TIGIT in T cells. tory molecule TIGIT and show that loss of TIGIT We show that loss of TIGIT results in hyperproliferative T cell responses and increased susceptibility to autoimmunity. in mice results in hyperproliferative T cell responses Furthermore, by generating an agonistic anti-TIGIT Ab, we http://www.jimmunol.org/ and increased susceptibility to autoimmunity. TIGIT demonstrate that TIGIT has T cell-intrinsic effects and that is thought to indirectly inhibit T cell responses by the signals through TIGIT directly inhibit T cell activation. induction of tolerogenic dendritic cells. By generating an agonistic anti-TIGIT Ab, we demonstrate that TIGIT Materials and Methods can inhibit T cell responses directly independent of APCs. Microarray analysis of T cells stimulated with Animals agonistic anti-TIGIT Ab revealed that TIGIT can C57BL/6-Tg(Tcra2D2,Tcrb2D2) (2D2) mice have been previously described 2/2 act directly on T cells by attenuating TCR-driven ac- (4). TIGIT mice were obtained from ZymoGenetics (Seattle, WA) and were generated by Ozgene (Bentley, Australia) using embryonic stem cells by guest on October 1, 2021 tivation signals. The Journal of Immunology, 2011, derived from C57BL/6 mice. C57BL/6 (B6) mice were purchased from the 186: 1338–1342. The Jackson Laboratory (littermate controls were used for at least one repeat of all of the experiments described, and results obtained did not differ from those using controls from The Jackson Laboratory), B6.SJL-Ptprca/BoAiTac (CD45.2 B6) mice were from Taconic (Hudson, NY), and Armenian ham- ecent genome-wide association scans have linked a sters were from Harlan Laboratories (Indianapolis, IN). Animals were kept in costimulatory molecule, CD226, to multiple auto- a conventional, pathogen-free facility at the Harvard Institutes of Medicine immune diseases in humans (1). CD226, together (Boston, MA), and all experiments were carried out in accordance with R guidelines prescribed by the Institutional Animal Care and Use Committee at with T cell Ig and ITIM domain (TIGIT), forms an emerging Harvard Medical School. pathway that has striking similarities to the well-known co- stimulatory CD28–CTLA-4 pathway. As in the B7-CD28– T cell activation and proliferation CTLA-4 pathway, CD226 and TIGIT bind the same set of Cells were cultured in DMEM with 10% FCS, 50 mM 2-ME, 1 mM sodium ligands (CD155 and CD112), and CD226 is a positive reg- pyruvate, nonessential amino acids, L-glutamine, penicillin, and streptomycin. + + ulator of T cell responses, whereas TIGIT inhibits them (2). A For in vitro T cell activation, CD4 and CD8 T cells were isolated using anti-CD4 or anti-CD8 beads (Miltenyi Biotec) and stimulated with plate- recent study suggested that TIGIT does not have any direct bound anti-CD3 (145-2C11, 2 mg/ml) and anti-CD28 (PV-1, 2 mg/ml) or effects on T cells, but instead acts on dendritic cells (DCs) soluble anti-CD3 (0.025 mg/ml) with irradiated splenocytes as APCs. Where

*Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical Swiss National Science Foundation and the Janggen-Po¨hn-Stiftung, J.P.H. is supported School, Boston, MA 02115; †Department of Medical Genetics, Juvenile Diabetes Re- by the Juvenile Diabetes Research Foundation and the Wellcome Trust, B.B. is a Swedish search Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Research Council Fellow, and S.S. is supported by Deutsche Forschungsgemeinschaft. Institute for Medical Research, Addenbrooke’s Hospital, University of Cambridge, ‡ Address correspondence and reprint requests to Dr. Vijay K. Kuchroo, Center for Cambridge, United Kingdom; Neurology Department, Yale Medical School, New x Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Haven, CT 06510; Medical and Population Genetics, Broad Institute of MIT and { HIM 785, Boston, MA 02115-5817. E-mail address: [email protected] Harvard, Cambridge, MA 02142; Department of Immunology, ZymoGenetics, Seattle, ‖ WA 98102; and Department of Pathology, Brigham and Women’s Hospital and Har- The online version of this article contains supplemental material. vard Medical School, Boston, MA 02115 Abbreviations used in this article: DC, dendritic cell; EAE, experimental autoimmune 1Current address: Novo Nordisk Inflammation Research Center, Seattle, WA. encephalomyelitis; GSEA, gene set enrichment analysis; IPA, ingenuity pathway analysis; KO, knockout; LN, lymph node; MOG, myelin oligodendrocyte glycoprotein; RT- Received for publication September 28, 2010. Accepted for publication December 6, PCR, real-time PCR; TIGIT, T cell Ig and ITIM domain. 2010. This work was supported by National Institutes of Health Grants P01AI056299, Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 P01AI039671, and R01NS035685 (to V.K.K. and A.H.S). N.J. is supported by the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003081 The Journal of Immunology 1339 indicated, cells were labeled with 2 mM CFSE. For costimulation with ago- II) . The microarray data are deposited at http://www.ebi.ac.uk/arrayexpress 2 nistic anti-TIGIT, CD4+MHC class II cells were sorted by flow cytometry with the accession number E-MEXP-2847. and stimulated with plate-bound anti-CD3 (0.5 mg/ml), anti-CD28 (0.5 mg/ ml), and anti-TIGIT (clone 4D4, 50 mg/ml) or isotype control (BioLegend). To determine proliferation, cells were pulsed with 1 mCi [3H]thymidine Results and Discussion (PerkinElmer) after 48 h and incubated for an additional 18 h before in- corporation was analyzed using a beta counter (1450 MicroBeta TriLux; TIGIT is expressed upon initial T cell activation PerkinElmer). TIGIT is upregulated on human T cells upon activation and Immunizations expressed on human memory and regulatory T cells (3). Where indicated, 105 TCR transgenic CD4+ T cells were transferred i.v. However, owing to lack of reagents, the expression of TIGIT 1 d prior to immunization. Mice were immunized s.c. with 100 mg myelin on mouse T cells has not been analyzed. To monitor TIGIT oligodendrocyte glycoprotein (MOG)35–55 peptide (MEVGWYRSPFSRV- surface expression, we generated a panel of TIGIT-specific 2 2 VHLYRNGK) emulsified in CFA. Organs were collected 7 or 8 d later, cells mAbs in TIGIT / mice and screened them for TIGIT speci- were restimulated with MOG35–55 peptide, and proliferation was determined by [3H]thymidine incorporation. Frequencies of MOG-specific cells were ficity. Clone 1G9 showed the best binding properties when determined after 5 d of restimulation with 30 mg/ml MOG35–55 peptide screened by ELISA (Supplemental Fig. 1A). It also specifi- b using MHC class II tetramers (I-A ) loaded with MOG35–55 or CLIP peptide cally stained TIGIT-transfected P815 cells and activated (PVSKMRMATPLLMQA, control) (20 mg/ml, 1.5 h at room temperature; National Institutes of Health Tetramer Core Facility, Atlanta, GA). Cytokine primary mouse T cells (Supplemental Fig. 1B,1C) as assessed concentrations in culture supernatants were determined by ELISA (IL-17) or by flow cytometry. We therefore used the 1G9 anti-TIGIT cytometric bead array (BD Biosciences, other cytokines). Ab to analyze the kinetics of TIGIT expression and compared Downloaded from Experimental autoimmune encephalomyelitis its expression to its costimulatory receptor CD226 in mouse T cells. Experimental autoimmune encephalomyelitis (EAE) was induced by s.c. TIGIT expression was induced upon stimulation, and immunization of mice with 10–15 mg of MOG35–55 peptide emulsified in CFA followed by 100 ng pertussis toxin (List Biological Laboratories) i.v. on mRNA levels steadily increased during the first 3 d of acti- + + day 0 and day 2, and classical clinical signs of EAE were scored as described vation in both CD4 and CD8 T cells (Fig. 1A). Inter- previously (4). Atypical signs were scored as 0.5 for each of the following: estingly, surface expression of TIGIT peaked at 24 h and dyskinesia, ataxia, and clasping phenotype. then decreased over time, even though mRNA levels kept http://www.jimmunol.org/ Generation of anti-TIGIT Abs increasing, suggesting that TIGIT expression was tightly reg- 2 2 ulated posttranscriptionally (Fig. 1B). This could be due to Armenian hamsters and TIGIT / mice were immunized with recombinant mouse TIGIT tetramers (ZymoGenetics) by a combination of s.c. and foot degradation and possibly also to receptor internalization, pad immunization and booster injections. Draining lymph nodes were fused which would functionally decrease surface TIGIT levels and with Sp2/0-Ag14 cells, selected in hypoxanthine/aminopterin/thymidine me- limit inhibitory signals at the initiation of the T cell response. dium, and supernatants were screened for specificity by ELISA and flow cyto- Despite a decrease in CD226 mRNA after 24 h, CD226 cell metry using TIGIT transfectants (ZymoGenetics). surface protein expression was transiently upregulated on Flow cytometry CD4+ T cells upon activation. Although CD8+ T cells con- by guest on October 1, 2021 Cells were stained in PBS, 0.1% sodium azide, and 0.5% BSA (20 min at 4˚C). stitutively express CD226 (8), T cell activation induced only Abs were from BioLegend, eBioscience (anti-Foxp3), BD Biosciences (7- minimal changes in CD226 mRNA and cell surface protein aminoactinomycin D), or they were generated as part of this study (anti- expression on CD8+ T cells (Fig. 1). TIGIT, clone 1G9). Samples were acquired on a FACSCalibur or LSR II 2 2 flow cytometer (BD Biosciences) and analyzed using the FlowJo software TIGIT / mice show augmented T cell responses upon immunization (Tree Star). 2 2 To begin to determine the role of TIGIT in vivo, TIGIT / Quantitative real-time PCR mice were immunized s.c. with MOG35–55, and the T cell RNA was extracted with RNAeasy Mini kits (Qiagen, Valencia, CA) and was analyzed by real-time PCR (RT-PCR) according to the manufacturer’s instruc- tions (Applied Biosystems, Carlsbad, CA). Primers–probe mixtures were: CD226 (Mm01301769m1), b-actin (Mm00446968-m1), TCRa (Mm01313019_g1), CD3« (Mm01179194_m1), phospholipase C-g1 (Mm01247293_m1), IL-2Rg (Mm00442885_m1), CD25 (Mm01340213_m1), and Bcl-xL (Mm00437- 783_m1). For TIGIT, primers and probe were: forward primer, 59-CTGATA- CAGGCTGCCTTCCT-39, reverse primer: 59-TGGGTCACTTCAGCTGT- GTC-39;probe,59-AGGAGCCACAGCAGGCACGA-39 (FAM, TAMRA).

Microarray Cells were harvested after 24 h stimulation, and RNA was isolated using RNeasy kits (Qiagen). GeneChip hybridization, staining, and scanning of the arrays were performed by the Partners HealthCare Center for Personalized Genetic Medicine (Cambridge, MA), according to the manufacturer’s in- structions (Affymetrix). Summarization of probe set intensity, background correction, and normalization was done using the Bioconductor imple- mentation of the GCRMA algorithm (5). Expression signals were compared using linear regression (6). In the single probe set analysis we used an a level $ # of 0.05 and regarded fold change 1.4 or 0.71 as significant. Ingenuity FIGURE 1. Expression of CD226 and TIGIT on T cells. CD4+ or CD8+ pathway analysis (Ingenuity Systems) was used to identify groups of genes or T cells were isolated from B6 mice and stimulated with plate-bound anti- pathways that show enrichment in significant molecules (fold change $1.2, p value of #0.1) and gene set enrichment analysis (GSEA) (7) to identify CD3 and anti-CD28. Samples were taken at the indicated time points and significant coordinate expression (using the Kyoto Encyclopedia of Genes and (A) expression of CD226 and TIGIT mRNA was determined by quantitative Genomes database, http://www.genome.jp/kegg). We created an additional RT-PCR (mean 6 SD). B, Surface expression of CD226 and TIGIT protein two pathways (see T cell activation and T cell survival in Supplemental Table was assessed by flow cytometry. 1340 CUTTING EDGE: T CELL INTRINSIC ROLE OF TIGIT response was analyzed 8 d later. When compared with B6 2 2 mice, T cells from TIGIT / mice displayed increased dose- dependent proliferation upon restimulation with antigenic peptide. T cell hyperproliferation was observed in draining 2 2 lymph nodes (LNs) and spleens of TIGIT / mice as well as in nondraining LNs, whereas no response was observed in the nondraining LNs of wild-type mice (Fig. 2A). Consistent with these results, tracking of Ag-specific CD4+ T cells with b 2/2 MOG35–55/I-A tetramers confirmed that TIGIT mice had higher frequencies of MOG-specific T cells in these organs (Fig. 2B). Similarly, MOG35–55-restimulated spleno- 2 2 2 2 cytes and LN cells from TIGIT / mice produced higher FIGURE 3. TIGIT / mice are more susceptible to EAE. A, B6 and 2/2 levels of proinflammatory cytokines, including IL-6, IFN-g, TIGIT mice were immunized s.c. with 10–15 mg MOG35–55 peptide in 2 2 and IL-17 (Fig. 2C). Interestingly, TIGIT / -derived cells CFA, followed by injection of 100 ng pertussis toxin i.v. on day 0 and day 2. Mice were monitored daily for EAE. Mean clinical score 6 SEM is shown, produced reduced basal levels of IL-10, which is in line with and linear regression curves of the disease for each group are depicted in the previous reports indicating that TIGIT induces IL-10 pro- inset (the 95% confidence intervals are represented with dashed lines; p , 2 2 duction in DCs (3). However, in addition to reduced basal 0.0001; n = 12/group). B, 2D2 TCR transgenic and 2D2 3 TIGIT / mice levels, IL-10 was also not induced by Ag-specific stimulation were monitored for the spontaneous onset of EAE. Mean clinical score 6

2 2 Downloaded from in TIGIT / cultures, suggesting that production of IL-10 by SEM is shown, and linear regression curves of the disease for each group are depicted in the inset (the 95% confidence intervals are represented with T cells is also impaired. In summary, these data demonstrate , that TIGIT acts as a negative regulator of T cell responses in dashed lines; p 0.0001). mice. 2 2 specific TCR transgenic mice (2D2 mice) (4). 2D2 mice do TIGIT / mice are more susceptible to EAE not develop spontaneous EAE but mostly develop spontane- Because CD226 has been genetically liked to susceptibility to ous optic neuritis. If TIGIT is an inhibitory molecule, we rea- http://www.jimmunol.org/ autoimmunity (1), and TIGIT, which shares the same ligands, soned that in the absence of TIGIT, 2D2 mice might develop 2 2 seems to act as an inhibitory molecule, we next tested wheth- EAE spontaneously. Indeed, 2D2 3 TIGIT / mice showed er the absence of TIGIT affects the development of auto- atypical signs of neurologic dysfunction as early as 28 d after 2/2 2 2 immunity. We immunized TIGIT and B6 mice for in- birth (Fig. 3B), and with advancing age all 2D2 3 TIGIT / duction of EAE with suboptimal doses of MOG35–55 (10–15 mice displayed dyskinesia, ataxia, and a clasping phenotype mg), such that B6 mice could not develop severe EAE but (9). Some of the 2D2 mice also showed these atypical symp- 2/2 TIGIT mice might display full-blown disease. Indeed, in toms. However, onset was strongly delayed and, in contrast 2/2 2/2 contrast to B6 mice, the vast majority of TIGIT mice to 2D2 3 TIGIT mice, 2D2 mice never progressed to by guest on October 1, 2021 developed severe EAE (Fig. 3A), supporting the notion of classical paralytic disease. These results emphasize the role of TIGIT as a coinhibitory molecule and suggesting an impor- TIGIT as a negative regulator of T cell responses and indicate tant role for the CD226–TIGIT pathway in regulating au- that TIGIT plays a role in limiting autoimmune responses, toimmune responses. and loss or dysfunction of this coreceptor may likely con- To assess the role of TIGIT in the control of spontaneous tribute to susceptibility to autoimmunity. 2/2 autoimmunity, we crossed the TIGIT mice to MOG35–55- TIGIT has T cell-intrinsic effects We next tested whether the augmented T cell responses dis- 2 2 played by TIGIT / mice were exclusively mediated through APCs or whether TIGIT can also directly affect T cells. To discriminate the effects mediated by APCs from those that are T cell intrinsic, we isolated CD4+ T cells from B6 and 2 2 TIGIT / mice, labeled them with CFSE, and stimulated them with either B6 or knockout (KO) APCs together with anti-CD3. Analysis of B6 T cell proliferation confirmed that TIGIT could mediate effects indirectly through APCs, as KO

2 2 APCs were better at promoting proliferation than were their FIGURE 2. TIGIT / mice show increased T cell responses upon immu- 2 2 wild-type counterparts (Fig. 4A, Supplemental Fig. 2A). nization. TIGIT / and B6 mice were immunized s.c. with 100 mgMOG 35–55 However, when wild-type APCs were used to stimulate B6 peptide in CFA. On day 8, spleens and LNs were harvested and cells were 2/2 2/2 3 restimulated with MOG35–55 peptide (0–100 mg, 10 dilution steps). A,Pro- or TIGIT T cells, the TIGIT T cells also showed liferation was measured after 48 h based on [3H]thymidine incorporation increased proliferation. The strongest proliferation was ob- (mean 6 SD, n =4).B, Tetramer staining was used to determine the fre- served when combining KO APCs with KO T cells, sug- + quencies of MOG35–55-specific CD4 T cells in spleens and LNs of B6 (filled gesting that TIGIT has synergistic roles on T cells and APCs. 2/2 squares) and TIGIT (open squares) mice. Dot plots from the draining LNs To dissect T cell-intrinsic and indirect effects of TIGIT as well as the frequencies of MOG35–55-specific cells of one representative 2/2 + + + in vivo, we transferred 2D2 or 2D2 3 TIGIT CD4 experiment are shown (percentage of tetramer cells within the live CD4 population, n =4).C, Cytokines were measured in the supernatants derived T cells into CD45.1 B6 hosts. These mice were immunized from the same cultures as in A at 48 h (n =4).*p # 0.05; **p # 0.001. s, with MOG35–55 and the T cell response was analyzed 7 d 2/2 spleen, d, draining LN; nd, nondraining LN. later. Despite comparable T cell expansion in vivo, TIGIT The Journal of Immunology 1341

A B coinhibitory molecules have the ability to dampen T cell acti- 2.5 p < 0.0001 spleen d LN nd LN vation, they differ in potency, kinetics of expression, and the 20 2.3 p = 0.0001 cellular pathways they alter. To understand how each coinhi- 2.1 100 1.9 bitory molecule alters T cell responses, it is critically important prolif. index prolif. -/- -/-

CPM (x1000) 0 to elucidate which signaling pathways are modulated by each of T cells: WT WT -/-TIGIT TIGIT-/- APC: WT WTTIGITTIGIT MOG: them. C D We used a whole genome microarray approach to identify γ the pathways affected by TIGIT engagement and compared 0.1 IL-63 IFN 4 IL-17 IL-10 15 isotype 1 4D4 + B6 -/--/- the gene expression in B6 CD4 T cells stimulated with ag- 2 TIGIT 10 2 2 2 / ng/ml 1 5 onistic anti-TIGIT 4D4 Ab to that of isotype and TIGIT 0 0 0 0 0 (x10’000) C.P.M. controls (Supplemental Table I). We found most differences MOG: - + - + - + - + - + - + - + - + - + - + - + - + 0 2.51 01 2.5 snddd snddsnddsnd αCD3[μg/ml] observed to be small, likely representing alterations of path- 2 2 FIGURE 4. TIGIT has T cell-intrinsic effects. Wild-type B6 or TIGIT / ways induced by T cell activation rather than separate path- CD4+ T cells were labeled with CFSE and stimulated with anti-CD3 in the ways that are induced through TIGIT (Supplemental Fig. 2/2 presence of irradiated B6 or TIGIT APCs. Proliferation was analyzed after 3A). Ingenuity pathway analysis (IPA) and GSEA (7, 13) were 60 h using flow cytometry, and proliferation indices were determined using used to identify pathways that are differently regulated if the FlowJo software (A)(n = 6). B and C, CD45.1 B6 recipients received 2 2 CD4+ 2D2 or 2D2 3 TIGIT / cells i.v. 1 d before s.c. immunization with TIGIT is engaged, and in line with the functional effects we MOG35–55 peptide. On day 7 spleens and LNs were harvested, cells were observed upon TIGIT activation, we found several pathways 3 Downloaded from restimulated with MOG35-55 peptide (0–100 mg, 10 dilution steps), and that are associated with T cell activation and cell cycle pro- proliferation was measured after 48 h ([3H]thymidine incorporation) (B)(n = gression to be enriched in the controls, indicating that TIGIT 4). C, Cytokines were measured in the supernatants derived from the same downregulates these pathways. An overlay of the microarray cultures as in B at 48 h using ELISA (IL-17) and cytometric bead array 2 2 data with the T cell activation pathway showed that a number (others; n = 4). D, CD4+ T cells were sorted from B6 and TIGIT / mice and stimulated with plate-bound anti-CD3 and anti-CD28 plus 4D4 or of molecules involved in TCR and CD28 signaling are sig- 3 nificantly downregulated upon TIGIT engagement and that

isotype control Ab. Proliferation was assessed by [ H]thymidine incorporation http://www.jimmunol.org/ (mean 6 SD). s, spleen; d, draining LN; nd, nondraining LN. many other key molecules involved, although not significantly decreased, show the same trend (Supplemental Fig. 4A). T cells showed increased proliferation upon restimulation Furthermore, we generated a gene set comprising key mole- with antigenic peptide and produced higher levels of proin- cules for the TCR and CD28 signaling pathways as well as flammatory cytokines (Fig. 4B,4C, Supplemental Fig. 2B). cell cycle progression and could verify that this gene set is 2 2 Importantly, 2D2 3 TIGIT / recipients also showed re- enriched in the control group using both GSEA and IPA duced IL-10 levels, confirming a T cell-intrinsic defect in (Supplemental Fig. 3B). Additionally, we have verified the 2 2 IL-10 production in TIGIT / T cells. TIGIT-mediated downregulation of three key molecules of To exclude any effects that might be due to conditioning of this pathway by RT-PCR (Supplemental Fig. 4C). TIGIT by guest on October 1, 2021 the APCs, we analyzed the T cell-intrinsic effects of TIGIT in seems to block productive T cell activation by directly acting an APC-free system. To completely eliminate any cells other on TCR expression itself, as engagement of TIGIT induced than T cells from our experiments, we tested our panel of a downregulation of the TCR a-chain as well as molecules TIGIT-specific Abs for functional agonistic activity in vitro. that comprise the TCR complex. Therefore, in contrast to Generating agonistic Abs directed against Ig superfamily other coinhibitory receptors (e.g., programmed death-1) that members, such as CD28, has proven to be a challenging interfere with processes that are further downstream in the endeavor, and when tested for functional effects, none of our TCR-induced signaling cascade (14), TIGIT acts upstream. Abs showed agonistic activity. We therefore generated a se- Although TIGIT engagement downregulated TCR activa- cond panel of TIGIT-specific Abs in Armenian hamsters and tion pathways, it did not inhibit cellular processes in general. screened them for specificity (Supplemental Fig. 2C–E) and We found expression of cytokine receptors that are associated functional activity. Out of these Abs, only one clone (4D4) with T cell maintenance (i.e., IL-2R, IL-7R, and IL-15R) as affected T cell proliferation in vitro. Clone 4D4 proved to be well as antiapoptotic molecules, such as Bcl-xL, to be upreg- agonistic, as the addition of plate-bound 4D4 to anti-CD3/ ulated by TIGIT (Supplemental Fig. 4B). As for the T cell anti-CD28–stimulated T cells inhibited their proliferation activation, we used RT-PCR to validate the differential ex- 2 2 (Fig. 4D). Importantly, when TIGIT / T cells were stim- pression of key genes of this pathway (Supplemental Fig. 4D) ulated with agonistic anti-TIGIT, no functional inhibition in and generated a core gene set for T cell maintenance and T cell proliferation was observed, confirming the specificity survival, which was found to be enriched in the TIGIT- of the Ab. Because no APCs were present in these in vitro stimulated sample using GSEA and IPA (Supplemental Fig. experiments, these data clearly demonstrate that TIGIT can 3C). In summary, TIGIT also inhibits T cell responses di- act directly on T cells. rectly by targeting molecules that are upstream in the T cell activation process while promoting T cell maintenance and TIGIT engagement modulates T cell activation survival; thus, although T cells are not activated and ex- Coinhibitory receptors on T cells are fundamental constitu- panded, the cells do not undergo anergy and are not deleted ents of the adaptive immune system necessary to limit T cell from the repertoire. responses to prevent chronic T cell activation, immunopathol- Our data support the idea that TIGIT is an inhibitory ogy, and autoimmunity, as demonstrated by the autoimmune- molecule, and loss of TIGIT in vivo increases T cell pro- 2 2 mediated lethality of the CTLA-4 / mice as well as the liferation, proinflammatory cytokine production, and accel- therapeutic approaches using CTLA-4-Ig (10–12). Although all erates development of autoimmunity. In addition to inhibiting 1342 CUTTING EDGE: T CELL INTRINSIC ROLE OF TIGIT

T cell responses via APCs, TIGIT can act directly on T cells, as 2. Tahara-Hanaoka, S., K. Shibuya, Y. Onoda, H. Zhang, S. Yamazaki, A. Miyamoto, S. Honda, L. L. Lanier, and A. Shibuya. 2004. Functional characterization of an agonistic anti-TIGIT Ab was able to inhibit T cell pro- DNAM-1 (CD226) interaction with its ligands PVR (CD155) and nectin-2 (PRR- liferation in the absence of any other cell type. Overall, our 2/CD112). Int. Immunol. 16: 533–538. results support the notion that TIGIT is an inhibitory mol- 3.Yu,X.,K.Harden,L.C.Gonzalez,M.Francesco,E.Chiang,B.Irving,I.Tom, S.Ivelja,C.J.Refino,H.Clark,etal.2009.ThesurfaceproteinTIGITsup- ecule that, in addition to affecting APC function, suppresses presses T cell activation by promoting the generation of mature immunoregu- T cell responses directly in a T cell-intrinsic manner. latory dendritic cells. Nat. Immunol. 10: 48–57. Although TIGIT deficiency per se does not result in the 4. Bettelli, E., M. Pagany, H. L. Weiner, C. Linington, R. A. Sobel, and V. K. Kuchroo. 2003. Myelin oligodendrocyte glycoprotein-specific T cell receptor induction of spontaneous autoimmunity or tissue inflamma- transgenic mice develop spontaneous autoimmune optic neuritis. J. Exp. Med. 197: tion, loss of TIGIT in a susceptible background can result in 1073–1081. 2 2 autoimmune disease as was observed in 2D2 3 TIGIT / 5. Wu, Z., R. A. Irizarry, R. Gentleman, F. Martinez-Murillo, and F. Spencer. 2004. A model-based background adjustment for oligonucleotide expression arrays. J. Am. mice, which spontaneously developed EAE. TIGIT might Stat. Assoc. 99: 909–917. therefore regulate the threshold of T cell activation and may 6. Smyth, G. K. 2004. Linear models and empirical bayes methods for assessing dif- be involved in the maintenance of peripheral tolerance. Fi- ferential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3: ar- ticle3. nally, the genetic linkage of the CD226–TIGIT pathway to 7. Subramanian, A., P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, M. A. a number of human autoimmune diseases (1) further indi- Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, and J. P. Mesirov. 2005. Gene set enrichment analysis: a knowledge-based approach for interpreting cates that this pathway plays an important role in limiting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102: 15545–15550. autoimmune responses, and loss or dysfunction of TIGIT 8. Dardalhon, V., A. S. Schubart, J. Reddy, J. H. Meyers, L. Monney, C. A. Sabatos, may likely contribute to susceptibility to autoimmune diseases. R. Ahuja, K. Nguyen, G. J. Freeman, E. A. Greenfield, et al. 2005. CD226 is

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