TIGIT Enhances Antigen-Specific Th2 Recall Responses and Allergic Disease Evangelia Kourepini, Nikolaos Paschalidis, Davina C

TIGIT Enhances Antigen-Specific Th2 Recall Responses and Allergic Disease Evangelia Kourepini, Nikolaos Paschalidis, Davina C. M. Simoes, Maria Aggelakopoulou, Jane L. Grogan and Vily This information is current as Panoutsakopoulou of October 2, 2021. J Immunol 2016; 196:3570-3580; Prepublished online 25 March 2016; doi: 10.4049/jimmunol.1501591 http://www.jimmunol.org/content/196/9/3570 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TIGIT Enhances Antigen-Speciﬁc Th2 Recall Responses and Allergic Disease

Evangelia Kourepini,* Nikolaos Paschalidis,* Davina C. M. Simoes,* Maria Aggelakopoulou,* Jane L. Grogan,† and Vily Panoutsakopoulou*

T cell Ig and ITIM domain receptor (TIGIT), expressed on T, NK, and regulatory T cells, is known as an inhibitory molecule that limits autoimmunity, antiviral and antitumor immunity. In this report, we demonstrate that TIGIT enhances Th2 immunity. TIGIT expression was upregulated in activated Th2 cells from mice with experimental allergic disease and in Th2 polarization cultures. In addition, its high-affinity ligand CD155 was upregulated in mediastinal lymph node dendritic cells from allergic mice. In an in vitro setting, we observed that Tigit expression in Th2 cells and its interaction with CD155 expressed in dendritic cells were important during the development of Th2 responses. In addition, blockade of TIGIT inhibited Th2, but had no effect on either Th1 or Th17 polarization. In vivo blockade of TIGIT suppressed hallmarks of allergic airway disease, such as lung Downloaded from eosinophilia, goblet cell hyperplasia, Ag-specific Th2 responses, and IgE production, and reduced numbers of T follicular helper and effector Th2 cells. Thus, TIGIT is critical for Th2 immunity and can be used as a therapeutic target, especially in light of recent findings showing TIGIT locus hypomethylation in T cells from pediatric patients with allergic asthma. The Journal of Immunology, 2016, 196: 3570–3580.

cell Ig and ITIM domain receptor (TIGIT), also known as allergic Th2 type responses remain unknown. Therefore, we WUCAM (1) or Vstm3 (2, 3), has been described as an addressed the role of TIGIT in Th2 immunity and specifically in http://www.jimmunol.org/ T inhibitory molecule expressed on memory T, activated T, Th2 recall responses that lead to allergic asthma. NK and regulatory T (Treg) cells (4–6). TIGIT has been found to A recent study demonstrated that the TIGIT locus is hypo- limit autoimmunity (2, 7) and to restrain T cell responses acting as methylated, and its gene product is overexpressed in CD4+ T cells an intrinsic inhibitory molecule by decreasing TCR activation, of pediatric patients with allergic asthma (14). In another study, proliferation, and cytokine secretion (5, 7). TIGIT expressed on T splenocytes of immunized Cd1552/2 mice secreted lower levels of or NK cells can bind to its high-affinity ligand CD155 (1), also IL-4 and contained fewer IL-4– and GATA-3–expressing CD4+ known as poliovirus receptor (8) or Necl-5 (9), expressed on the T cells (15). Of note, a recent report showed that TIGIT-expressing + surface of APCs (4, 10, 11). Engagement of TIGIT to CD155 Foxp3 T cells could not suppress allergic asthma upon transfer by guest on October 2, 2021 promotes IL-10 while it restrains IL-12 production by dendritic (6). The above findings suggest that TIGIT may play a role in Th2 cells (DCs), leading to reduced T cell activation in vitro (4). immunity, possibly distinct from its role in other types of immu- + TIGIT-expressing Foxp3 T cells have been recently characterized nity. In this report, we provide evidence that TIGIT expression by as a highly functional Treg cell subset that selectively suppresses Th cells and its interaction with CD155 enhances Th2 responses, Th1 and Th17 responses (6). In addition, upregulated TIGIT ex- and blockade of TIGIT is therapeutic for experimental allergic + pression by tumor-infiltrating CD8 T cells promotes T cell ex- airway inflammation. haustion and tumor expansion (12, 13). Likewise, high TIGIT + expression by CD8 T cells inhibits antiviral immunity (12). Al- Materials and Methods though TIGIT expression by T cells has suppressive effects on Mice autoimmunity, antiviral, and antitumor immunity, its effects on BALB/c and OVA-specific TCR-transgenic DO11.10 (Tcr-TG-DO11.10) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). *Cellular Immunology Laboratory, Center for Basic Research, Biomedical Research Mice were housed at the Animal Facility of the Biomedical Research Foundation of the Academy of Athens, Athens 115 27, Greece; and †Department of Foundation of the Academy of Athens. Protocols were approved by the Cancer Immunology, Genentech, South San Francisco, CA 94080 Bioethics Committee of Biomedical Research Foundation of the Academy ORCIDs: 0000-0002-2299-1007 (E.K.); 0000-0002-1569-1508 (V.P.). of Athens and the Greek Government. All procedures were in accordance with the National Institutes of Health Statement of Compliance (Assur- Received for publication July 17, 2015. Accepted for publication February 23, 2016. ance) with Standards for Humane Care and Use of Laboratory Animals This work was supported by the European Research Council (ERC) under the Eu- (A5736-01) and with the European Union Directive 86/609/EEC for the ropean Union’s Seventh Framework Programme (FP/2007–2013)/ERC Grant Agree- protection of animals used for experimental purposes. ment 243322 (to V.P.) and by the Greek General Secretariat of Research and Technology Grant Aristeia I 2618 (to V.P.). In vivo experimental protocols Address correspondence and reprint requests to Dr. Vily Panoutsakopoulou, Biomed- ical Research Foundation of the Academy of Athens, 4 Soranou Efessiou Street, Mice were immunized with 0.01 mg chicken OVA (Sigma-Aldrich) in 0.2 Athens 115 27, Greece. E-mail address: [email protected] ml aluminum hydroxide (alum; Serva) i.p. on days 0 and 12. After the two The online version of this article contains supplemental material. immunizations (on day 18), draining lymph nodes (LNs) of DO11.10 mice were isolated, and CD4+CD44hiCD62L+CD127+ T memory cells were Abbreviations used in this article: alum, aluminum hydroxide; BAL, bronchoalveolar sorted for cocultures with naive DCs. For allergic airway disease induction lavage; DC, dendritic cell; LN, lymph node; MLN, mediastinal lymph node; PAS, periodic acid–Schiff; PD-1, programmed cell death-1; Tfh, T follicular helper; after OVA/alum immunizations, BALB/c mice were administered aero- TIGIT, T cell Ig and ITIM domain receptor; Treg, regulatory T. solized OVA (5%, for 20 min) three times on days 18–20. Each mouse received 20 mg affinity-purified blocking polyclonal Ab against TIGIT Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 (AF-7267, R&D Systems) or corresponding Ig isotype control Ab (R&D www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501591 The Journal of Immunology 3571

Systems) three times i.p., 2 h before challenges. Similarly, we used 20 mg/ Quantitative PCR mouse purified monoclonal blocking Abs against TIGIT (1G9), CD155 RNA extraction, quantification, and cDNA synthesis from isolated DCs or (4.24.1-LEAF), or isotype control Ig Abs (MOPC-21 and RTK2758- + LEAF; BioLegend). For certain experiments, we administered 250 mg/ CD4 T cells were performed as previously described (20). Primers were mouse monoclonal IgG2a blocking Ab against TIGIT (clone 10A7) (4, designed using the Primer 3 program and are as follows: Tigit sense primer, 12), kindly provided by Genentech, or the corresponding Ig isotype control 59-GTGGGATTTACAAGGGGAGA-39 and antisense, 59-CTTCCAGGG- Ab (R&D Systems). Mice were euthanized on day 21. We stained paraffin- GATGAGAGAC-39; Gata3 sense primer, 59-TTACCACCTATCCGCCC- embedded lung sections (4 mm) with H&E and periodic acid–Schiff (PAS) TAT-39 and antisense, 59-ACACACTCCCTGCCTTCTG-39; Cd226 sense to evaluate lung infiltration and goblet cell hyperplasia, as previously de- primer, 59-CAAGAACACTGGCACAAAGA-39 and antisense, 59-GAAA- scribed (16). We used a semiquantitative scoring system to grade the size CAAGCAGGAGTAGATGC-39; c-maf sense primer, 59-CCCTTGA- of lung infiltrates as previously described (17). Goblet cells were counted CAGTTTGCTTCTA-39 and antisense, 59-CCCATTCTGCTATCTTTGAC-39; on PAS-stained lung sections using an arbitrary scoring system as de- Rorc sense primer, 59-TGTTTTATGGGGTTTGGGTA-39 and antisense, scribed previously (17). Images from H&E and PAS stained sections were 59-AAGAGATTGTGTGCCAGAG-39;andTbx21 sense primer, 59- obtained by a Leica DM LS2 optical microscope and analyzed with Leica TAGTGATTGGTTGGAGAGGA-39 and antisense, 59-GTAGTTCGGGCA- Application Suite V3.6.0 (Leica Microsystems). We harvested, measured, GAGAAAG-39 (Eurofins MWG). Primers for Cd155 were previously de- and analyzed bronchoalveolar lavage (BAL) inflammatory cells, as pre- scribed (15). Real-time PCR was performed as previously described (20). viously described (16, 17). Serum Ab concentration of OVA-specific IgE was measured with an ELISA kit (BioLegend). We used a previously Statistical analysis described method to isolate cells from draining LNs (18). Mediastinal Data were analyzed using Prism Software (GraphPad) and the unpaired LNs (MLNs) from immunized or asthmatic mice were harvested, Student t test. The p values #0.05 were considered significant. pooled from each mice group, and stimulated ex vivo with 125 mg/ml OVA (Sigma-Aldrich). Measurements of cytokine levels were performed in culture supernatants, as previously described for LN cells (18). MLN CD4+ T cells were isolated with the CD4+ T Cell Isolation Results Downloaded from Kit (Miltenyi Biotec). TIGIT on Th cells and its ligand CD155 on DCs are overexpressed under Th2 conditions Cultures and cytokine analysis Initially, we polarized isolated naive Th cells toward Th1, Th2, Draining LNs from immunized or naive mice were harvested. Naive T cells were isolated from BALB/c mice (for Th polarization) and naive or or Th17 and analyzed their TIGIT expression. We observed that memory T cells from DO11.10 mice (for cocultures) with the CD4+ Th2-polarized cells harbored the most significant increase in CD62L+ T Cell Isolation Kit (Miltenyi Biotec). FACS sorting (FACSAria Tigit expression (Fig. 1A). As expected, newly differentiated http://www.jimmunol.org/ III; BD Biosciences) followed for further purification with anti-mouse Th1 and Th17 cells had also upregulated expression of Tigit mAbs: CD3 (145-2C11), CD4 (GK1.5), CD62L (MEL-14), CD44 (IM7), (Fig. 1A). However, after reactivation, only cells of the Th2 CD25 (PC61), and CD127 (A7R34; BioLegend). DCs were enriched + with the CD11c microbead kit (Miltenyi Biotec) and FACS sorting fol- subset upregulated Tigit expression (Fig. 1B). Moreover, TIGIT lowed with anti-mouse mAbs for: CD19 (6D5) and CD11c (N418). For Th2 cells were higher in percentages when compared with Th1 dead cell exclusion, 7-aminoactinomycin D was used (BD Biosciences). and Th17 cells (Fig. 1C). Effective polarization was verified by The two-step coculture was previously described by Alpan et al. (19). In the relevant expression of signature transcription factors Gata3, our setting, isolated DCs from LNs and spleens of naive mice were cocultured either with T memory cells (Th2) from two times DO11.10- Tbx21, and Rorc (Supplemental Fig. 1A) and IL-4, IL-13, IL-5, IFN-g, and ΙL-17 cytokine secretion for each subset (Supplemental

sensitized mice (OVA/alum) or with T cells from naive DO11.10 mice, as by guest on October 2, 2021 control, for 12 h in the presence of 100 mg/ml OVA in a cell ratio of 1:35 Fig. 1B). Highly purified naive CD4+ T cells used in these ex- DC/T. After 12 h, Th2- or T naive–activated DCs were sorted and periments were CD252, and no Foxp3 expression was detected cocultured again for 72 h with naive DO11.10 T cells in the presence of before and after Th2 differentiation/reactivation (data not 100 mg/ml OVA in a ratio 1:350 DC/T cells. Blocking Abs against TIGIT (1G9), CD155, or isotype control Ig Abs (BioLegend) were used in a shown). concentration of 10 mg/ml in cocultures. For Th polarization cultures, We then investigated whether TIGIT expression in the Th2 anti-CD3/28 beads (Life Technologies) were added for 96 h together cultures could influence their ability to produce lineage-specific with 20 ng/ml rIL-2 (PeproTech) for Th0, 20 ng/ml rIL-2, 20 ng/ml rIL- cytokines. We found that TIGIT+ outnumbered the TIGIT2 IL- 12 (PeproTech), and 10 mg/ml purified anti–IL-4 (BioLegend) for Th1, ∼ 20 ng/ml rIL-2, 20 ng/ml rIL-4 (PeproTech), 10 mg/ml anti–IFN-g 4– and IL-5–expressing cells in the Th2 cultures (4- and 2-fold, (BioLegend) for Th2, and 10 ng/ml rTGF-b, 100 ng/ml rIL-6 (Pepro- respectively) (Fig. 1D). In contrast, in the rest of the Th cell Tech), 10 mg/ml anti–IFN-g, and anti–IL-4 for Th17. To restimulate cultures, we noticed significantly less TIGIT+ compared with polarized Th cells, new anti-CD3/28 beads were added for 72 h. Com- TIGIT2 IFN-g–expressing cell numbers in the Th1 (Fig. 1E), and mercially available ELISA kits were used for detection of mouse IL-4, accordingly less TIGIT+ IL-17–expressing cells in the Th17 IL-13, IL-5, IL-10, IFN-g, and IL-17 (R&D Systems) in all culture su- 2 pernatants and BAL fluid. culture, as measured to the TIGIT fraction (Fig. 1F). Thus, the levels of TIGIT expression were elevated among Th2- Flow cytometry differentiated cells that produced high amounts of IL-4 and IL- Freshly isolated MLN cells were stained with combinations of 5, but no difference was observed in Th1 or Th17 cell cultures. fluorochrome-conjugated Abs to CD3, CD4, CD44, CD62L, CD11c, Moreover, sorted TIGIT+ Th2 was the cell subpopulation that TIGIT (1G9), CD155 (TX56), IL-13Ra1 (SS12B), T1ST2 (DIH9), ICOS expressed significantly higher Gata3 and cmaf levels at 72 h of (C398.4A), programmed cell death-1 (PD-1; 29F.1A12) (BioLegend), differentiation (Fig. 1G). Although significantly higher percent- and CXCR5 (L138D7) (BD Pharmingen). Before intracellular staining, + + 2 CD16/32 mAb (BioLegend) was used to block FcgRII/III receptors. ages of TIGIT Th2 cells were IL-4 compared with the TIGIT Intracellular cytokine expression was assessed by 25 ng/ml PMA (Sigma- (Fig. 1H), in IL-5+ cells, percentages were similar among TIGIT+ Aldrich) and 1 mg/ml ionomycin calcium salt (Sigma-Aldrich) for a 5-h and TIGIT2 Th2 cells (Fig. 1H). Accordingly, we measured high incubation and a Cytofix/Cytoperm Kit Plus (Golgiplug; BD Biosciences). levels of IL-4 and IL-5 secreted by TIGIT+ Th2 cells in the po- For intracellular cytokine staining, cells were stained with surface markers 2 and then with Abs against IL-4 (11-B11), IL-5 (TRFK5), IL-13 (eBio13A), larization culture (data not shown). Nevertheless, TIGIT Th2 hi + IL-17A (TC11-18H10.1), IFN-g (XMG1.2), and IL-10 (JES5-16E3) (eBio- were activated cells (CD44 ), similarly to TIGIT Th2 (data not science and BioLegend). For proliferation assays, naive CD4+ Tcells shown), and capable of producing lineage-specific cytokines. (Fig. from BALB/c spleens were stained with CellTrace Violet (Life Tech- 1D, 1H). nologies) and activated with anti-CD3/28 beads for 96 h. All stained samples were analyzed in Attune Acoustic Focusing Cytometer (Applied Basedontheseresults,wethenasked whether allergic mice Biosystems), and the raw data were calculated and visualized with upregulated TIGIT. We found significantly increased numbers of FlowJo Software (Tree Star). TIGIT+ CD4+ T cells in draining mediastinal LNs (MLNs) from 3572 TIGIT ENHANCES Th2 RESPONSES Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 1. Elevated expression of TIGIT on Th2 cells. (A)RelativeTigit expression of differential Th cell subsets after in vitro polarization (96 h). Relative Tigit expression of restimulated polarized Th cell subsets from (A) with anti-CD3/28 (72 h) (B), and representative FACS plots with percentages of TIGIT+ Th1, Th2, and Th17 cells after restimulation (C). Numbers of IL-4+ and IL-5+CD4+ T cells in Th2 cultures (D), IFN-g+ CD4+ T cells in Th1 cultures (E), and IL-17+CD4+ T cells in Th17 cultures (F) after 96 h of differentiation. Relative Gata3 and c-maf expression (G) and percentages of IL-4+ and IL-5+TIGIT2 and TIGIT+ subpopulations (H) in Th2 cultures (96 h). Data are expressed as mean 6 SEM and are from two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.0002. mice with Th2-mediated allergic airway inflammation compared We then activated naive DCs with Th2 memory cells in the with sensitized control mice (Fig. 2A). Allergic mice also presence of Ag in vitro. Th2 memory cells were isolated from LNs exhibited higher percentages of TIGIT+ CD4+ T cells in MLNs of OVA-sensitized mice. Upon activation, Th2 memory cells compared with sensitized mice, presenting both effector CD44hi expressed high levels of Tigit (Fig. 3A). We also observed sig- CD62L2CD1272 CD4+ T and CD44hiCD62L+CD127+ central nificantly elevated Cd155 mRNA expression in DCs cocultured memory CD4+ T cells (Fig. 2A). In support, MLN CD4+ T cells with Th2 memory cells (Fig. 3A). In contrast, DCs cocultured from allergic mice expressed higher levels of Tigit mRNA (Fig. 2B), with naive CD4+CD442CD62L+ T cells had almost undetectable and a substantial percentage of TIGIT+CD4+CD44hi T cells were levels of Cd155 expression (Fig. 3A), indicating that the sig- expressing IL-4 (Fig. 2C). Of note, the TIGIT+ IL-4–expressing nificant elevation of Cd155 expression in DCs is correlated with T cells of allergic mice were also T1ST2+ (data not shown). the higher Tigit expression in Th2 memory cells compared with Certain reports show that CD155, the high-affinity ligand of Th naive cells. Subsequent coculture of these CD155-expressing TIGIT, is expressed on T cells upon their activation (5) and can DCs with naive T cells in the presence of Ag led to IL-4, IL-5, mediate a costimulatory signal on CD4+ T cells (21). We did not and IL-13 cytokine secretion (Fig. 3B). Th cultures induced with observe any difference in CD155+ CD4+ T cell numbers and CD155-expressing DCs expressed significantly higher levels percentages (Fig. 2D) derived from allergic MLNs compared with of Gata3 when compared with the cultures in which DCs had CD4+ T cells from sensitized mice. In contrast, we found that DCs minimal CD155 expression (Fig. 3C). However, the expression derived from MLNs of allergic mice were CD155+, and their levels of both Tbx21 and Rorc remained the same (Fig. 3C). numbers were significantly elevated compared with DCs from Following their coculture with Th2-activated DCs, the newly sensitized mice that were mostly CD1552 (Fig. 2E). Percentages differentiated Th2 cells exhibited also significantly increased of CD155+ DCs from MLNs of allergic mice were also elevated Tigit expression (Fig. 3C). In contrast, DCs that expressed low compared with sensitized mice (Fig. 2E). These findings are in Cd155 did not induce high Tigit expression in new Th2 cells agreement with studies that demonstrate elevated TIGIT expres- (Fig. 3C). Expression of Cd226, the alternative CD155 ligand in sion by activated T cells and CD155 upregulation by activated Th2 cells, had no difference between the two DC groups DCs (4, 5, 15, 22, 23). (Fig. 3C). Thus, levels of Tigit expression on newly differenti- The Journal of Immunology 3573 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Elevated expression of TIGIT on T cells and of CD155 ligand on DCs in MLNs of allergic mice. (A) Numbers of MLN TIGIT+CD4+ Tcellsof allergic (yellow) compared with sensitized mice (white) and representative FACS plots with percentages of MLN TIGIT+ T cells of allergic versus sensitized + + hi 2 + + hi + mice, gated on CD3 CD4 CD44 CD62L effector T (Teff) cells and CD3 CD4 CD44 CD62L T central memory (TCM)cells.(B)RelativeTigit expression in MLN CD4+ T cells of sensitized and allergic mice. (C) Percentages of IL-4+ cells within the CD44hiT1ST2+TIGIT+CD4+ MLN T cell subpopulation. Numbers, representative FACS plots with percentages of MLN CD155+ DCs (D) and CD155+CD4+ T cells of allergic versus sensitized mice (E). Data are expressed as mean 6 SEM. n = 6–8 mice per group. All data are from two independent experiments. ***p , 0.0002. 7AAD, 7-aminoactinomycin D. ated Th2 cells (Fig. 3C) are correlated with levels of Cd155 an enhancer for Ag-speciﬁc Th2 cells in recall responses. We expression on DCs (Fig. 3A). Conclusively, the above results measured Ag-speciﬁc Th2 cytokine secretion in the presence or suggest that TIGIT on Th cells and its ligand CD155 on DCs are absence of a polyclonal TIGIT-blocking Ab (Fig. 4A) in Th2 cell highly abundant under Th2 conditions. differentiation cultures, in which activated DCs (by Th2 memory cells) were added. Interestingly, TIGIT blockade restrained IL-4, The TIGIT–CD155 interaction is crucial for Th2 induction by IL-13, IL-5, IL-10, and IFN-g secretion by newly differentiated DCs in vitro Th2 cells compared with Ig Ab (Fig. 4A). Inhibition of TIGIT TIGIT has been generally described as inhibitory molecule for binding also restrained Gata3 expression in newly differentiated T cell responses (4–6). We thus questioned whether elevated Th2 cells, whereas Tbx21 and Rorc expression remained in the TIGIT expression in Th2 cells could possibly act as an inhibitor or same levels (Fig. 4B). 3574 TIGIT ENHANCES Th2 RESPONSES Downloaded from

FIGURE 3. Th2-activated DCs that express CD155 induce new Th2 differentiation. (A) Relative Tigit expression in DO11.10 Th2 memory cells after their 12-h coculture with naive DCs and OVA and Cd155 mRNA expression in DCs (yellow). Control culture with naive DO11.10 T cells, DCs, and OVA (white). Levels of IL-4, IL-13, and IL-5 secretion (B) and relative Gata3, Tbx21, Rorc, Tigit, and Cd226 mRNA expression (C) in DO11.10 Th cells cocultured with Th2-activated DCs (yellow) or DO11.10 T naive-activated DCs (white) for 72 h in the presence of OVA. Data are mean 6 SEM from three independent experiments. ***p , 0.0002. http://www.jimmunol.org/

Similarly to TIGIT blockade, use of a CD155-blocking Ab had in Th1 and Th17 cell polarization cultures had no effect on inhibitory effects on Ag-speciﬁc Th2 cytokine secretion (Fig. 4C), TIGIT+/TIGIT2 IFN-g– (Fig. 5C) or TIGIT+/TIGIT2 IL-17– as well as on Gata3 expression (Fig. 4D). Furthermore, inhibition expressing cell numbers, respectively (Fig. 5D). These results of TIGIT or CD155 binding also restrained Tigit expression in suggest that TIGIT blockade could effectively attenuate Th2 cell newly differentiated Th2 cells (Fig. 4B, 4D), indicating a possible polarization also in the absence of DCs, but has no apparent en- feedback loop regulation. In the same cultures, blockade of either hancing effect on Th1 and Th17 differentiation. TIGIT or CD155 caused upregulation of Cd226 expression, the TIGIT blockade upon allergen challenge protects from allergic

alternative ligand of CD155, compared with Ig control Abs by guest on October 2, 2021 airway disease (Fig.4B, 4D). Moreover, by blocking concomitantly both TIGIT and CD155, we found even lower secretion of IL-4 and IL-13 As TIGIT is highly expressed by activated Th2 cells (Figs. 1B, 2B, when compared with cultures in which either TIGIT or CD155 2C), and CD155 is upregulated in both Th2-activated DCs was blocked (Supplemental Fig. 2A). This dramatic attenuation of (Fig. 3A) and in DCs from allergic mice (Fig. 2E), we sought to Th2 activity upon concomitant inhibition of TIGIT and CD155 is investigate the potential role of TIGIT in the in vivo Th2-driven possibly explained by the fact that there is no other interaction responses and allergic airway disease. We thus administered a favored. polyclonal purified blocking Ab against TIGIT in mice developing Therefore, TIGIT blockade mediates significant downregula- allergic asthma during allergen (OVA) challenges. Blockade of tion of Gata3 expression in the Th2 setting, implying that TIGIT TIGIT conferred significant suppression of disease, with de- signaling maintains Th2 responses (Figs. 4B, 4D, 5B). Impor- creased histological score (Fig. 6A) and lower numbers of BAL tantly, neither TIGIT Ab nor CD155 Ab, when compared with Ig eosinophils and macrophages, compared with mice treated with control Abs, elevated IFN-g secretion that could lead to hampered the control Ig Ab (Fig. 6B). Importantly, serum OVA-specific IgE Th2 responses through a switch to Th1 (Fig. 4A, 4C). In different responses were significantly decreased in TIGIT Ab-treated mice settings, TIGIT ligation enhanced IL-10 production by T cells (5, (Fig. 6C). 7, 24), which was consistent with our observation of reduced IL- During the effector phase of an allergic immune response, Ig 10 secretion upon either TIGIT or CD155 blockade (Fig. 4A, 4C). production depends on the help provided by T follicular helper cells Overall, TIGIT–CD155 interaction is crucial for Th2 induction by (Tfh) to B cells, and Tfh cells are important for IgE-mediated DCs upon Ag-specific recall responses in vitro. responses in allergic disease (25). Also, Tfh cells can be derived from Th2-committed cells after antigenic challenge in vivo (26) TIGIT blockade inhibits Th2 but not Th1 and Th17 polarization and can be IL-4 producers (27, 28). TIGIT was firstly identified in We also blocked TIGIT in cultures of naive Th cells that were Tfh cells that express elevated TIGIT compared with naive CD4+ differentiated in Th2 cells through TCR stimulation and in the T cells (1, 29). TIGIT blockade resulted in significantly decreased absence of DCs. Upon blockade, numbers of both TIGIT+/TIGIT2 numbers of CXCR5+ICOS+PD-1+ Tfh MLN cells (Fig. 6D), IL-4– and IL-5–expressing cells were significantly reduced consistent with the observed inhibition of Ag-specific IgE pro- (Fig. 5A). As expected, TIGIT Ab caused reduction in Gata3 duction (Fig. 6C). expression of Th2-differentiated cells (Fig. 5B). TIGIT blockade Moreover, TIGIT blockade restrained the numbers of total CD4+ in the polarization cultures also affected the cell numbers of the CD44hiCD62L2CD1272 effector T cells with inflammatory Τh2 TIGIT2 IL-4– and IL-5–secreting populations, indicating that phenotype (T1ST2+, IL-4+, IL-13Ra1+) in MLNs (Fig. 6D). More possibly a soluble factor produced by TIGIT+ cells important for specifically, TIGIT blockade limited the numbers of MLN effector Th2 cell differentiation is inhibited. In contrast, TIGIT blockade T1ST2+ and/or IL-13Ra1+CD4+ effector T cells, indicating a re- The Journal of Immunology 3575

FIGURE 4. TIGIT–CD155 interaction is crucial for Th2 differentiation upon recall responses. Levels of IL-13, IL-4, IL-5, IL- 10, and IFN-g (A) and relative Gata3,

Tbx21, Rorc, Tigit, and Cd226 mRNA ex- Downloaded from pression (B) in DO11.10 Th cells after 72 h of coculture with Th2-activated DCs, OVA, and TIGIT Ab (orange) or Ig control Ab (white). Cytokine levels (C) and relative Gata3, Tigit, and Cd226 mRNA expression (D) of DO11.10 Th cells after 72 h of coculture with Th2-activated DCs, OVA, http://www.jimmunol.org/ and CD155 Ab (orange) or Ig control Ab (white). Data are mean 6 SEM from ﬁve independent experiments. *p , 0.05, **p , 0.01, ***p , 0.0002. by guest on October 2, 2021

duction in IL-33– and IL-13–induced signaling, crucial for aller- and Tfh) in MLNs (Fig. 7C), and reduced CD4+ T cell Gata3 gic asthma (30, 31). IL-10+CD4+ effector T cells were also de- expression (Fig. 7D). 1G9 TIGIT Ab also dampened IL-4, IL-13, creased (Fig. 6D). IL-5, and IL-10 cytokine production in culture supernatants of TIGIT blockade also led to significantly lower levels of Gata3 Ag-specific responses of MLNs (data not shown). expression in MLN CD4+ T cells (Fig. 6E). Accordingly, in vivo In order to exclude the possibility that the specific TIGIT Abs TIGIT blockade caused significantly dampened IL-4, IL-13, IL-5, we use for blockade exert any agonistic activity on T cells, we and IL-10 cytokine production in culture supernatants of Ag- performed proliferation assays, as previously described (2, 5, 7). specific responses of MLNs (Fig. 6F), whereas no effect on Neither of the Abs was able to inhibit proliferation of anti- IFN-g and IL-17 secretion was observed (Fig. 6F). Similarly, BAL CD3/28–stimulated CD4+ T cells (Supplemental Fig. 2B, 2C). fluid from lungs of mice treated with TIGIT Ab had lower levels of In fact, upon addition of 1G9, proliferation remained the same IL-4, IL-13, and IL-5. In contrast, IL-10 and IFN-g levels had no (Supplemental Fig. 2B), whereas the use of the polyclonal significant differences compared with BALs of Ig Ab-treated mice TIGIT Ab enhanced the proliferative capacity of CD4+ Tcells (Fig. 6G). BAL fluid had undetectable levels of IL-17 (not shown). (Supplemental Fig. 2C). Thus, neither of these two Abs pre- We also observed disease suppression when we used a mono- sented any agonistic activity. clonal blocking Ab against TIGIT (1G9) in vivo. Treatment with To further support our findings, we administered a widely 1G9 Ab resulted in decreased histological score (Fig. 7A), lower tested mAb against TIGIT (10A7) with established blocking serum Ag-specific IgE production (Fig. 7B), decreased numbers of capacity (4, 12). The results obtained were in agreement with proinflammatory CD4+ effector T cells (T1ST2+, IL-4+, IL-13R+, previous findings when the polyclonal and 1G9 TIGIT Abs 3576 TIGIT ENHANCES Th2 RESPONSES

FIGURE 5. TIGIT enhances Th2 differentiation also in the absence of DCs. Num- bers of TIGIT+ and TIGIT2 among IL-4+ and IL-5+CD4+ T cells (A) and relative Gata3 expression after 96 h in Th2 polarization cultures (B). (C and D) Numbers of TIGIT+ and TIGIT2 among IFN-g + and IL- 17+CD4+ T cells after 96 h in Th1 and Th17 polarization cultures, respectively. TIGIT Ab or Ig Ab was added in the beginning of all Th cultures (A–D). Data are mean 6 SEM, from two independent experiments. *p , 0.05, ***p , 0.0002. Downloaded from

were used (Fig. 6, 7). In fact, upon Ag challenges, mice ad- Discussion http://www.jimmunol.org/ ministered with blocking Ab 10A7 exhibited significantly re- In agreement with previous reports showing TIGIT expression in duced histological scores, mucus secretion (Supplemental Fig. activated CD4+ T cells (4, 5), we initially observed that upon 3A), and decreased numbers of BAL cells compared with mice stimulation, newly differentiated Th2 cells, as well as memory treated with the control Ig Ab (Supplemental Fig. 3B). Im- Th2 cells, expressed high levels of TIGIT. Moreover, elevations in portantly, serum Ag-specific IgE responses were significantly TIGIT expression were specifically confined to the Th2 subset decreased in TIGIT Ab-treated mice (Supplemental Fig. 3C). upon reactivation. Secondary stimulation of Th2 cells resulted in Moreover, numbers of MLN CD44hiCD4+ T cells (T1ST2+,IL- dramatically higher TIGIT expression, whereas this was not ap- 13+,IL-4+,IL-5+,IL-10+, and Tfh) were also lower compared parent for Th1 and Th17 cells. In vivo, mice with Th2-driven + hi + by guest on October 2, 2021 with controls, whereas the population of IFN-g CD44 CD4 allergic asthma presented high levels of TIGIT expression in a T cells had no difference (Supplemental Fig. 3D). 10A7 dampened high proportion of effector T and central memory CD4+ T cells IL-4 and IL-13 production in culture supernatants of Ag-specific derived from MLNs. As TIGIT on Th cells interacts with ligands responses of MLNs (Supplemental Fig. 3E), as well as IL-4, IL- expressed on APCs (4), we asked whether blockade of these in- 13, and IL-5 production in BAL fluid (Supplemental Fig. 3F). teractions could have an effect on Th2 immunity. TIGIT blockade In contrast, 10A7 administration had no effect on Ag-specific restrained DC and Ag-driven Th2 responses in vitro. Treatment IFN-g and IL-17 secretion in MLNs and BAL (Supplemental Fig. using two different types of blocking TIGIT Abs suppressed Ag- 3E, 3F). specific Th2 responses and IgE production, as well as eosinophilia Conclusively, TIGIT blockade upon antigenic challenge sig- and the development of allergic airway disease. Use of the mono- nificantly limited Th2 responses and, consequently, protected from clonal blocking TIGIT Ab 10A7 also reduced goblet cell hyper- allergic asthma. In the context of Th2 recall responses, in vivo plasia and, consequently, mucus production. Thus, although TIGIT TIGIT blockade did not enhance Th1 and Th17 Ag-specific re- signaling appears to be inhibitory for Th cells and for autoimmu- sponses (Fig. 6F, Supplemental Fig. 3D). nity (2, 7, 32), our findings pointed to a proallergic, Th2-enhancing role for this molecule. CD155 blockade upon allergen challenge suppresses Th2 TIGIT ligand CD155 expression in follicular DCs is important responses for effective Ab secretion (1, 29), and Cd1552/2 mice have re- Consistent with the in vitro blockade of CD155 (Fig. 4C, 4D), duced Tfh cell numbers as well as defective development of when we used the same blocking Ab against CD155 in vivo, secondary humoral immune responses (29, 33). We observed that, allergic mice showed decreased Th2 responses. Specifically, similar to anti-TIGIT, blocking of the TIGIT ligand CD155 during serum Ag-specific IgE-production (Fig. 7F), numbers of proin- antigenic challenge resulted in significant suppression of Th2 re- flammatory CD4+ effector T cells (T1ST2+,IL-4+,IL-13Ra1+, sponses. A previous report showed that Cd1552/2 mice exhibited andTfh)inMLNs(Fig.7G),andCD4+ TcellGata3 expression decreased percentages of splenic IL-4+GATA3+CD4+ T cells upon were significantly reduced (Fig. 7H) in allergic mice adminis- OVA/CpG immunization (15). We also observed that blocking of tered with the CD155 Ab compared with those that received the either CD155 or TIGIT resulted in dampened Ag-specific IgE Ig control Ab. Nevertheless, CD155 blockade could not confer production, decreased proinflammatory T1ST2+ or IL-13Ra1+ significant protection from lung infiltration, as the unaltered Th2 and Tfh cell numbers in MLNs, as well as reduced Gata3 histological score between CD155 Ab- and Ig Ab-treated mice expression in MLN CD4+ T cells. These results indicated the revealed (Fig. 7E). Therefore, CD155 Ab administration upon significance of the TIGIT–CD155 interaction in the Th2 context. antigenic challenge could limit Th2 responses in a fashion In contrast, a recent study showed that primary OVA immuniza- similar to TIGIT Abs. tion of Cd1552/2 mice resulted in increased percentages of splenic The Journal of Immunology 3577 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. TIGIT blockade with a polyclonal Ab upon allergen challenge protects mice from allergic airway disease. (A) Photomicrographs and score of lung inflammation from BALB/c allergic mice treated three times with either TIGIT Ab (orange) or Ig control Ab (white) upon OVA challenges. Scale bars, 100 mm. BAL differentials (B), OVA-specific IgE serum levels (C), and numbers of MLN CD3+CD4+CD44hiCD62L2 effector T cells (D) presenting T1ST2+, IL-4+, IL-13Ra1+, IL-10+, and CXCR5+ICOS+PD-1+ Tfh cells per mouse. (E) Relative Gata3 mRNA expression in isolated MLN CD4+ T cells of allergic mice. Data from one representative experiment. Levels of IL-4, IL-13, IL-5, IL-10, IFN-g, and IL-17 in supernatants of OVA-stimulated MLN cells (F) and levels of IL-4, IL-13, IL-5, IL-10, and IFN-g in BAL fluid (G). Data are expressed as mean 6 SEM; n = 5–7 mice per group (A–G), three independent experiments (A–D, F, and G). *p , 0.05, **p , 0.01, ***p , 0.0002. Eos, eosinophils; LMs, lymphocytes and monocytes; Macs, macrophages; Neuts, neutrophils.

IL-4– and IL-13–secreting CD4+ T cells and higher total serum Ig its blockade resulted in reduced IgE-dependent activation of production compared with wild-type (21). However, immune pa- mast cells (36). Thus, interruption of TIGIT–CD112 interaction rameters were only measured after primary sensitization and not could be inducing reduced lung eosinophilia, observed in TIGIT following secondary Ag responses. In addition, although the blockade, but not in CD155 blockade experiments. Clarification of authors mentioned that Cd1552/2 mice had exacerbated airway the possible role of CD112 in this context could provide new inflammation, the changes in eosinophilia were modest, and Ag- insights in allergy. In addition to TIGIT expressed on T cells, specific responses were not measured (21). Our findings indicated TIGIT Ab blocks TIGIT on other cell types that mediate allergic that CD155 has an enhancing role for peripheral Th2 responses responses (37). For example, TIGIT is highly expressed on NK during antigenic challenge. The use of a CD155-blocking Ab (34, cells and is an inhibitor of NK cell cytotoxicity (3, 38, 39). 35) in our study provided a clearer view of its significance as a However, the role of TIGIT in NK2 cells that mediate asthma is clinical target for airway inflammation. However, although anti- unknown and merits investigation (40). Overall, TIGIT–CD155 TIGIT inhibited eosinophilia, lung infiltration, and goblet cell interaction appears to be very important for Th2 cell responses in hyperplasia, anti-CD155 did not have a significant effect on these the allergic model. The possibility of TIGIT interactions with features of allergy. It is possible that interactions of TIGIT with other ligands cannot be excluded that would be important for other molecules could mediate an important role for lung infil- eosinophilia and lung cell infiltration. tration. TIGIT blockade suppressed all known hallmarks of allergic CD155 blockade inhibits only TIGIT–CD155 interaction, airway inflammation. For these experiments, we used a TIGIT- whereas TIGIT Ab blocks the interaction of TIGIT with other specific polyclonal Ab previously shown to effectively block ligands, such as CD112 (Nectin-2) (2, 4). CD112 is highly TIGIT in vitro (38). We alternatively tested a mAb against TIGIT expressed on eosinophils mediating mast cell degranulation, and (1G9) that had no agonistic activity (7). We confirmed that both 3578 TIGIT ENHANCES Th2 RESPONSES

FIGURE 7. TIGIT or CD155 blockade upon allergen challenge protects mice from allergic airway disease. (A) Score of lung inflammation from BALB/c allergic mice treated three times with either 1G9 TIGIT Ab (blue) or Ig control Ab (white) upon OVA challenges. OVA-specific IgE serum levels (B) and numbers of MLN CD3+CD4+ CD44hiCD62L2 effector T cells (C) that are T1ST2+, IL-4+, IL-13Ra1+, and of CXCR5+ ICOS+PD-1+ Tfh cells per mouse. (D) Rel- ative Gata3 mRNA expression in isolated MLN CD4+ T cells of allergic mice. Data from one representative experiment. Data are expressed as mean 6 SEM; n = 5 mice A D per group ( – ), three independent experi- Downloaded from ments (A–C). (E) Score of lung inflammation from allergic mice treated three times with either CD155 Ab (orange) or Ig control Ab (white) upon OVA challenges. OVA-specific IgE serum levels (F) and numbers of MLN CD3+CD4+CD44hiCD62L2 effector T cells + + + (G) that are T1ST2 , IL-4 , IL-13Ra1 , and http://www.jimmunol.org/ of CXCR5+ICOS+PD-1+ Tfh cells per mouse. (H) Relative Gata3 mRNA expression in isolated MLN CD4+ T cells of allergic mice. Data from one representative experiment. Data are expressed as mean 6 SEM; n = 5 mice per group (A and B), two independent experiments (A–C). *p , 0.05, **p , 0.01, ***p , 0.0002. by guest on October 2, 2021

Abs do not exert agonistic activity. In our experiments, 1G9 Ab allergic disease, and this was explained by reduced Fgl2 expres- almost completely inhibited all aspects of allergic response; sion by these cells (6). It is very likely that in vivo blockade of however, the reduction in histological score and the Ag-specific TIGIT has an effect on both Treg and Th2 cells. Our preliminary IgE production were not as dramatic as the ones induced by the experiments showed that suppression of allergic disease by either polyclonal TIGIT Ab. The polyclonal Ab targets several epitopes TIGIT or CD155 Ab was correlated with reduction in levels of of TIGIT molecule, and this could be the reason of the described Fgl2 expression in MLN cells (data not shown). Whether down- differences in efficacy. The blocking ability of both Abs (poly- regulation of Fgl2 expression is confined to Treg and/or Th2 cells clonal and 1G9) were in agreement with the findings obtained by remains to be elucidated. administration of the monoclonal blocking TIGIT Ab 10A7, Previously, T memory–DC–Th cell crosstalk was described to demonstrating disease attenuation. In fact, administration of 10A7 mediate oral tolerance (19). Our in vitro experiments demon- showed more effective dampening of allergic inflammation that strated this axis to be of high importance in the context of Th2 led to significant reduction of goblet cell hyperplasia and mucus secondary responses. Actually, we showed that DCs, after condi- secretion. tioning by Th2 memory cells, can promote differentiation of new Moreover, TIGIT activation in Th cells using an agonistic Th cells toward Th2, and this important DC–Th crosstalk was TIGITAbhasbeenfoundtorestrainIFN-g secretion (5) and regulated by the CD155–TIGIT interaction. CD155-expressing enhance Il10 gene expression (6). Our experiments using blocking DCs were highly efficient for induction of TIGIT-expressing nonagonistic Abs for TIGIT in vivo led to unaltered levels of IFN-g, Th2 cells. In agreement with Yu et al. (4) showing that TIGIT as well as downregulated IL-10 secretion in MLNs. expressed on human memory T cells interacts with CD155 A previous report described a TIGIT-expressing Foxp3+ Treg expressed on DCs, our data demonstrated for the first time, to our cell subset (6). TIGIT expression by these cells renders them knowledge, that CD155 expressed on activated DCs induces suppressive in autoimmunity but not in allergic disease upon TIGIT expression on newly differentiated Th2 cells. As expected, transfer (6). The explanation provided for the lack of efficacy of TIGIT operated as a Th2 cell enhancer in vitro, similarly to the TIGIT+ Treg cells in the Th2 context was that these cells produce in vivo Th2 response. Fgl2 that is contributing to allergic responses (41). In addition, Also, TIGIT blockade in Th2 differentiation cultures without DC TIGIT2Foxp3+ Treg cells were suppressive for Th2-mediated addition inhibited Th2 cell polarization. In contrast, a previous The Journal of Immunology 3579 study demonstrated that the use of a TIGIT agonistic Ab in human be an explanation for the Th2-enhancing activity of TIGIT we memory T cell cultures exhibited an inhibitory role of TIGIT found upon recall allergic responses. Moreover, microarray anal- signaling for T-bet, Rorc, and Gata3 expression (5). In that study, ysis revealed that although TIGIT engagement downregulated the authors used human memory CD4+ T cells and performed Th2 TCR expression, this was not inhibiting for several other impor- cultures without IFN-g blockade, whereas Gata3 expression was tant cellular processes (7). In fact, TIGIT promoted T cell main- measured at an early time point (5). Moreover, blockade of a re- tenance and survival by driving expression of several cytokine ceptor, compared with its agonistic triggering, does not always receptors (IL-2R, IL-7R, and IL-15R) and antiapoptotic molecules lead to opposite effects. For example, deficiency of TIGIT ex- (7). These findings explain the capacity of TIGIT to promote pression by T cells was reported to significantly inhibit IL-10 (5, expression of activating T cell molecules and are in no contrast to 7), whereas agonistic activation of TIGIT failed to increase their our results that indicate enhancement of Th2 cell activation upon IL-10 production (5). These results are consistent with our find- recall responses. ings in which TIGIT blockade had no apparent enhancing effect Overall, in this study, we demonstrate that TIGIT has a stim- on Th1 and Th17 differentiation, whereas agonistic TIGIT trig- ulatory role in Th2 responses and allergic disease, which is distinct gering has inhibitory effects upon certain conditions (5). from its inhibitory role in autoimmunity and antitumor immunity Furthermore, we found another CD155 ligand, Cd226 (or (2, 7, 13). Our findings suggest a direct effect of TIGIT on Th2 DNAM-1) receptor to be expressed in newly differentiated Th2 responses, pointing to TIGIT as a potential therapeutic target for cells. Cd226 expression was elevated in Th2 cells in vitro upon asthma. These results may also enhance our understanding of disruption of CD155–TIGIT interaction. Blocking of the CD155– antitumor immunity as TIGIT blockade is used to boost effective TIGIT interaction led, apart from dampening Tigit expression, to responses in cancer (12, 13). Importantly, in the Th2 context, we enhanced CD226 expression. A recent study described CD226/ observed decreased levels of IFN-g secretion upon TIGIT/CD155 Downloaded from CD155 interaction on the T cell surface to be Th2 suppressing, blockade in vitro and stable IFN-g and IL-17 secretion upon as it downregulates IL-4, IL-13 levels, and GATA3 expression in TIGIT blockade in vivo. Therefore, we demonstrated that block- human nonpolarized T cells via enhancement of Th1 and Th17 ade of TIGIT to lower Th2 responses is a selective strategy that responses (42). Thus, in our setting, if activation of CD226 by does not enhance concomitant Th1 and Th17 responses, which are CD155 was inhibitory for Th2 responses, then blockade of either very important for maintenance of antiviral, antibacterial, and of these molecules would lead to Th2 enhancement. However, antifungal immunity. http://www.jimmunol.org/ we observed exactly the opposite, as blockade of CD155 limited Th2 responses and IFN-g production. In addition, simultaneous Acknowledgments blockade of both TIGIT and CD155 had similar effects to either We thank Genentech for kindly providing the blocking 10A7 Ab. We TIGIT or CD155 blockade and dampened Th2 responses. Thus, it also thank M. Bessa, F. Gargoulas, I. Skordos, E. Chala, M. Tzioras, is unlikely that interaction of CD226 with CD155 could restrain E. Christakou, and E. Papaioannou for technical assistance. Th2 responses in our setting. Supportively, a previous study reported that Th2 differentiation was not regulated through CD226 Disclosures stimulation (43), and importantly, CD226 blockade had no effect J.L.G. is employed by Genentech, a corporation that develops and markets by guest on October 2, 2021 on Th2 polarization (42). drugs for profit. The other authors have no financial conflicts of interest. The ITIM domain of TIGIT expressed on NK cells was found to be responsible for inhibition of their cytotoxicity (3). This observation led to the current belief that TIGIT could also act as an References 1. Boles, K. S., W. Vermi, F. Facchetti, A. Fuchs, T. J. Wilson, T. G. Diacovo, inhibitory molecule for T cells, through its ITIM domain, although M. Cella, and M. Colonna. 2009. A novel molecular interaction for the adhesion there is no proof for its functionality in T cells. A study, which of follicular CD4 T cells to follicular DC. Eur. J. 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A Gata3 Tbx21 Rorc

B IL-17 IL-4 IL-13 IL-5 IFN-γ /ml g p

1 2 7 1 2 7 1 2 7 h h 1 h h 1 h h 1 T T h T T h T T h T T T

Supplemental Figure 1. Effective polarization was verified in Th cultures. (A) Relative Gata3, Tbx21, Rorc expression and (B) IL-4, IL-13, IL-5, IFN-γ, IL- 17 secretion of re-stimulated Th cells. Data presented are from one representative experiment.

1 Supplemental Figure 2

A Ag-specific responses:

IL-4 IL-13 Ig Ab TIGIT Ab *** *** *** ** CD155 Ab *** ** *** * *** *** *** *** TIGIT+ CD155 Ab

pg/ml

B C T cells + anti-CD3/28 T cells + anti-CD3/28 Day Specimen0: control 14 1G9 Bl6.fcs 63 DaySpecimen 0: control 12 TIGIT Ab Bl6.fcs 81.7 FSC-A, SSC-A subset Specimen 15 isotype Bl6.fcs 62.5 Specimen 15 isotype Bl6.fcs 100 VL1-A, FSC-A subset 86% Day Specimen4: Ig Ab 11 stained unstimulated.fcs 67%39.8 DaySpecimen 4: Ig Ab 11 stained unstimulated.fcs 39.8 VL1-A, FSC-A subset 85% Day 4: 1G9 Ab 83% Day 4: TIGIT Ab % of %Max % of %Max

Cell Trace Cell Trace

Supplemental Figure 2. TIGIT-CD155 interaction is crucial for Th2 differentiation upon recall responses. (A) Levels of IL-13, IL-4 of DO11.10 Th cells after 72h of co-culture with Th2-activated DCs, OVA and 1G9 TIGIT Ab (orange), CD155 Ab (orange with lines), TIGIT and CD155 together (brick red) or Ig control Ab (white). Data are expressed as mean ± S.E.M. and are from two independent experiments *p<0.05, **p<0.01, ***p<0.0002. (B) Proliferation of naïve CD4+ T cells stimulated with anti-CD3/28 and treated with 1G9 TIGIT Ab (green line) or Ig control Ab (blue line) for 96h, or (C) polyclonal TIGIT Ab (green line) or Ig control Ab (blue line) treated. Data presented are from one representative experiment.

2 Supplemental Figure 3

A B C OVA-specific IgE: 50

/ml) 15 4 40 e r

o *

c 30 10 ** s Ig Ab S 20 ** ng/ml A **

P 5 ** 10 ** TIGIT Ab (10Α7) ** BALcells (x10 0 0

Eos LMs D Macs Neuts MLN CD44hi CD4+ T cells:

+ + + + + + T1ST2 IL-13 IL-4 IL-5 IFN-γ IL-10 TFH ) 4 40 16 20 6 20 30 *** 10 ** *** ** 20 8 *** 10 3 10 15 *** 5

0 0 0 0 0 0 0 Cells per mouse (x10

E Ag-specific responses:

IL-4 IL-13 IFN-γ IL-17

** ** 0 350 700 0 2500 5000 0 2500 5000 0 300 600

pg/ml

F BAL supernatants:

IL-4 IL-13 IL-5 IFN-γ IL-17

* ** *** 0 400 800 0 300 600 0 100 200 0 200 400 0 125 250

pg/ml

Supplemental Figure 3. TIGIT blockade upon allergen challenge protects mice from allergic airway disease. (A) Histological score of lung inflammation and mucus secretion from BALB/c allergic mice treated 3x with either 10A7 Ab (blue) or Ig control Ab (white) upon OVA challenges. (B) BAL differentials, (C) OVA-specific IgE serum levels and, (D) numbers of MLN CD3+CD4+CD44hi T cells presenting T1ST2+, IL-13+, IL-4+, IL-5+, IFNγ+, IL-10+ and CXCR5+ICOS+PD-1+ Tfh cells per mouse. (E) Levels of IL-4, IL-13, IFN-γ and IL-17 in supernatants of OVA stimulated MLN cells and, (F) IL-4, IL-13, IL-5, IFN-γ and IL-17 levels in BAL fluid. Data are expressed as mean ± S.E.M. n=5 mice per group, two independent experiments. *p<0.05, **p<0.01, ***p<0.0002.