Smad and NFAT Pathways Cooperate to Induce CD103 Expression in Human CD8 T Lymphocytes

Smad and NFAT Pathways Cooperate To Induce CD103 Expression in Human CD8 T Lymphocytes

This information is current as M'Barka Mokrani, Jihène Klibi, Dominique Bluteau, of September 24, 2021. Georges Bismuth and Fathia Mami-Chouaib J Immunol 2014; 192:2471-2479; Prepublished online 29 January 2014; doi: 10.4049/jimmunol.1302192

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Supplementary http://www.jimmunol.org/content/suppl/2014/01/28/jimmunol.130219 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 56 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/192/5/2471.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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Smad and NFAT Pathways Cooperate To Induce CD103 Expression in Human CD8 T Lymphocytes

M’Barka Mokrani,*,†,‡ Jihe`ne Klibi,*,†,‡,1 Dominique Bluteau,x,2 Georges Bismuth,{,‖,# and Fathia Mami-Chouaib*,†,‡

The interaction of integrin aE(CD103)b7, often expressed on tumor-infiltrating T lymphocytes, with its cognate ligand, the epithelial cell marker E-cadherin on tumor cells, plays a major role in antitumor CTL responses. CD103 is induced on CD8 T cells upon TCR engagement and exposure to TGF-b1, abundant within the tumor microenvironment. However, the transcriptional mechanisms underlying the cooperative role of these two signaling pathways in inducing CD103 expression in CD8 T lymphocytes remain unknown. Using a human CTL system model based on a CD8+/CD1032 T cell clone specific of a lung tumor–associated Ag, we demonstrated that the transcription factors Smad2/3 and NFAT-1 are two critical regulators of this process. We also identified promoter and enhancer elements of the human ITGAE gene, encoding CD103, involved in its induction Downloaded from by these transcriptional regulators. Overall, our results explain how TGF-b1 can participate in CD103 expression on locally TCR- engaged Ag-specific CD8 T cells, thus contributing to antitumor CTL responses and cancer cell destruction. The Journal of Immunology, 2014, 192: 2471–2479.

ytotoxic T lymphocytes are major effector cells of the transplantation (5) and in promoting renal allograft rejection (6, 7).

immune system, predominantly responsible for Ag-specific They are also involved in selective destruction of pancreatic http://www.jimmunol.org/ C clearance of tumors and infected cells. These effector cells islet allografts (8) and host intestinal epithelium during graft- exert their lytic activity following interaction of TCR with the versus-host disease (9–12), which can be prevented by CD103 specific peptide–MHC class I complex on target cells, mainly through deficiency (13). The aEb7 integrin is expressed at high levels by exocytosis of cytotoxic granules. Upon initial TCR-dependent target mucosal CD8+ T lymphocytes, in particular intestinal epithe- cell recognition, adhesion/costimulatory molecules, including lium lymphocytes (14), psoriatic skin epidermal CD8+ T cells integrins, are repositioned at the T cell–target cell contact zone (15), and cervicovaginal Ag-specific CTL (16). It is also found referred to as the immune synapse. This results in the formation of on mucosal mast cells and dendritic cells (17), CD4+ and CD8+ a signaling complex with intracellular proteins and the initiation T regulatory (Treg) cells (18, 19), and on a large proportion of of a transduction cascade, leading to execution of CTL effector CD8+ effector T cells infiltrating epithelial tumors, including by guest on September 24, 2021 functions, mainly killing of target cell and cytokine secretion. bladder (20), colorectal (21), pancreatic (22), ovarian (23), and lung Among integrin family members, the aE(CD103)b7 integrin cancers (1, 3). The restricted distribution of the aEb7 integrin is plays an essential role in TCR-mediated cancer cell lysis by inter- attributed to expression of the aE subunit (CD103), because the b7 acting with its ligand, the epithelial cell marker E-cadherin, on tu- subunit is widely distributed on T lymphocytes (24). mor cells, triggering the release of cytotoxic granules by specific It is now widely admitted that CD103 can be induced on CD8 + + CTL (1–4). CD8 /aEb7 T lymphocytes have been reported to play T cells residing in tissue microenvironments in which TGF-b1is a critical role in mediating tubular injury following allogeneic renal abundant, including epithelia, chronic inflammatory lesions (25), and tumors (26, 27), or during autoimmune processes (28) and renal allografts (6, 29, 30). Indeed, accumulating evidence indi- *INSERM U753, F-94805 Villejuif, France; †Gustave Roussy, 94805 Villejuif Cedex, cates that TGF-b1 is directly involved in CD103 induction upon x France; ‡Universite´ Paris-Sud, 91400 Orsay, France; INSERM U1009, Gustave T cell activation (31, 32) and that its regulation occurs at the Roussy, 94805 Villejuif Cedex, France; {INSERM U1016, Institut Cochin, 75014 Paris, France; ‖Centre National de la Recherche Scientifique, UMR8104, 75014 Paris, transcription level (33, 34). However, little is known about the France; and #Universite´ Paris Descartes, 75006 Paris, France molecular mechanisms that regulate expression of the ITGAE gene, 1Current address: Pasteur Institute, Paris, France. which encodes CD103. A proximal promoter region has been pre- 2Current address: Laboratoire De´veloppement du Syste`me Immunitaire, Ecole Pra- viously described, but it did not confer TGF-b1 responsiveness, tique des Hautes Etudes, Institut Universitaire d’He´matologie, Saint-Louis Hospital, suggesting the existence of distal control elements (34). In this re- Paris, France. port, we demonstrate that Smad and NFAT pathways cooperate to Received for publication August 20, 2013. Accepted for publication December 23, induce ITGAE gene expression in CD8 T cells. We also identify 2013. promoter and enhancer regulatory elements of the human ITGAE This work was supported by grants from INSERM, the Association pour la Re- cherche sur le Cancer (Grant SFI20111203599), and Institut National du Cancer, gene with effective Smad3 and NFAT-1 binding sites whose part- Projet Libre. nership activates CD103 expression and antitumor cytotoxicity of Address correspondence and reprint requests to Dr. Fathia Mami-Chouaib, INSERM CD8 T cells after TCR engagement in the presence of TGF-b1. U753, Team 1: Tumor Antigens and T-Cell Reactivity, Gustave Roussy 114, Rue E´douard Vaillant, F-94805 Villejuif, France. E-mail address: [email protected] The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CsA, cyclo- T cell clone and tumor cell lines sporin A; siRNA, small interference RNA; Treg, T regulatory. The CD1032 T cell clone H32-22 was isolated from PBL of a patient Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 suffering from a nonsmall cell lung carcinoma (35). This clone recognizes, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302192 2472 ROLE OF Smad AND NFAT IN ITGAE GENE EXPRESSION on the autologous tumor cell line IGR-Heu, a mutated a–actinin-4 epitope Construction of ITGAE promoter and enhancer reporter presented in a HLA-A2 context (36). plasmids and luciferase assay The IGR-Heu tumor cell line was established from the nonsmall cell lung carcinoma patient tumor, as previously described (36). Human leukemic ITGAE promoter and enhancer sites were predicted by Genomatix software CD8 T cell line, TALL-104, was purchased from the European Collection (http://www.genomatix.de). A 899-bp fragment of the ITGAE promoter of Cell Culture. The 293-T cells were obtained by transformation of human and a 843-bp fragment of the ITGAE enhancer were amplified by PCR 293 embryonic kidney cells by the large T Ag from the SV40 virus. Jurkat- from genomic DNA of the autologous CD103+ T cell clone Heu171 (1) Tag T cells, stably transfected with the SV40 large T Ag, were derived and cloned into the pGL4.12 vector (Promega) between SacI and XhoI from the human leukemia CD4 T cell line Jurkat. sites. Primer pairs used for amplification were as follows: promoter forward, 59-ATAGAGCTCCATCGCCACTCTGCACTTCCAGCAGCC-39 and Ab, chemical inhibitors, and flow cytometry analysis promoter reverse, 59-ATACTCGAGCATCCTTGCTGGAGCAGAGGCG- 9 9 mAb directed against CD8 and CD103 were purchased from Immunotech or GCTGTG-3 ; enhancer forward, 5 -ATAGGATCCCATTTACATGGGGT- 9 9 eBiosciences. Anti-CD3 (UCHT1) mAb were provided by BD Biosciences. CTTGTTCTGTCACCCAGG-3 and enhancer reverse, 5 -ATAGTCGAC- 9 Anti-Smad2, antiphosphorylated (phospho) Smad2, anti-Smad3, anti–phospho- CATGAGCAAGGATATAGAAGATATGAACAAC-3 . We then cloned the Smad3, anti–NFAT-1, and negative control mAb were purchased from Cell ITGAE enhancer fragment into a BamHI site located downstream of the Signaling Technology. Anti-actin and secondary mAb were purchased from luciferase gene in the ITGAE promoter reporter plasmid. Smad site dele- Santa Cruz Biotechnology. tion mutants were created from this reporter plasmid by site-directed TGF-b type 1 receptor (TGR-bR1) kinase inhibitor SB-431542 (SB) mutagenesis using a mutagenesis kit (Quickchange II XL Site-Directed was purchased from Sigma-Aldrich. Smad3 inhibitor SIS3 and calcineurin Mutagenesis Kit; Agilent Technologies) and the following primer pairs: 9 inhibitor cyclosporin A (CsA) were provided by Calbiochem. promoter forward, 5 -CGAGGACCCTGGGCTGGAAGCTGTTCTGAGG- 9 9 Phenotypic analyses of T lymphocytes were performed by direct im- CAGGACAGGG-3 and reverse, 5 -CCCTGTCCTGCCTCAGAACAGCT- 9 9 munofluorescence using a FACSCalibur flow cytometer. Data were pro- TCCAGCCCAGGGTCCTCG-3 ; enhancer forward, 5 -GCACCACTGC-

9 Downloaded from cessed using CellQuest software (BD Biosciences). ACTCCAGCCTGGTGACGTCTCAAAAAAAAAAGAAAAATG-3 and 9 For CD103 induction, T cells were prestimulated with a combination of reverse, 5 -CATTTTTCTTTTTTTTTTGAGACGTCACCAGGCTGGAG- 9 anti-CD3 mAb (UCHT1 at 5 mg/ml) coated to plastic plates and rTGF-b1 TGCAGTGGTGC-3 . (5 ng/ml; Abcys) (1), in the absence or presence of the inhibitors, and ex- Luciferase assays were performed in 293-T cells or Jurkat-Tag T cells. A 3 5 m pression of CD103 was assessed at day 4, which corresponds to the optimal total of 2 10 cells was transfected with 1 g luciferase reporter plasmid time point for CD103 expression (data not shown). and 100 ng Renilla luciferase control plasmid using jetPRIME Polyplus transfection and then cultured for 17 h at 37˚C in six-well flat-bottom cell RNA interference culture plates (Corning Life Sciences). Transfected cells, untreated or treated with the TGF-bR1 kinase inhibitor SB, were either kept in medium or http://www.jimmunol.org/ Gene silencing of Smad2 expression in the H32-22 T cell clone was per- stimulated with TGF-b1. Cells were then collected and analyzed for luciferase formed using predesigned Smad2 small interference (si)RNA (siRNA-S2) activity by the dual-luciferase reporter assay system (Promega). All assays purchased from Ambion (catalogue 4392420). Briefly, cells were trans- were repeated at least three times, and the activity of firefly luciferase (pGL4) fected by electroporation with 1 mM siRNA in an Amaxa system using V was normalized to that of the internal control, Renilla luciferase (pRL-CMV). solution and X-001 program. Luciferase siRNA, siRNA-Luc (siRNA duplex, 59-CGUACGCGGAAUACUUCGAdTdT-39,and59-UCGAAGUAUUCCG- Chromatin immunoprecipitation assay CGUACGdTdT-39), included as a negative control, was purchased from Sigma-Proligo. Chromatin immunoprecipitation (ChIP) assay was performed with the H32- 22 T cell clone, unstimulated or stimulated for 30 min with a combination Cytotoxicity assay and Western blot analysis of anti-CD3 mAb and rTGF-b1 using the SimpleChIP enzymatic ChIP kit by guest on September 24, 2021 The cytotoxic activity of the T cell clone was measured by a conventional (Cell Signaling). Cells were fixed (1% formaldehyde) for 10 min at room 4-h 51Cr-release assay using triplicate cultures (1). The autologous tumor temperature, chromatin was sheared by sonication, and lysates were clarified cell line, IGR-Heu, was used as a target cell at indicated E:T cell ratios. by centrifugation. Immunoprecipitating mAb (anti-Smad2, anti–phospho- Inhibition of lysis was assessed by preincubating effector cells with indi- Smad3, anti–NFAT-1, or anti-IgG negative control), validated for the ChIP cated inhibitors for 1 h at room temperature. assay, were then added to the cross-linked chromatin preparation and in- For Western blot analysis, T cells (5 3 106) were either kept in medium cubated at 4˚C overnight. Immune complexes were collected by addition or stimulated for 30 min with anti-CD3 mAb (UCHT1), TGF-b1, or of protein G magnetic beads, washed, and eluted by spin columns. To a combination of both reagents in the absence or presence of SB (5 mM) determine the percentage of each analyzed region against the input DNA, or SIS3 (5 mM) inhibitors, or pretreated with siRNA-S2. Total cellular quantitative RT-PCR was performed using Power SYBR Green PCR Master extracts were obtained by cell lysis in ice-cold lysis buffer (10 mM HEPES Mix (Applied Biosystems). PCR oligonucleotide primers were as follows: Smad binding site on ITGAE promoter sense, 59-GGAGGCACTTCCT- [pH 7.4], 150 mM NaCl, 1% CHAPS, 1% glycerol) supplemented with 9 9 9 a mixture of antiproteases (Roche) and orthovanadate (2 mM) for 30 min ATGGGAG-3 and antisense, 5 -ATCATCCATTTGAGTCGGTCC-3 ;NFAT m binding site on ITGAE promoter sense, 59-GAACGCAGCCGAAGTT- at 4˚C. Equivalent amounts of protein extracts (20 g) were denatured in 9 9 9 Laemmli buffer, separated by SDS-PAGE on 4–20% precise protein gel, GAATC-3 and antisense, 5 -TCTTGTACCAGCCCATGCAC-3 ;Smadand NFAT binding sites on ITGAE enhancer sense, 59-CAGTCAGCCAAGAT- and transferred onto a nitrocellulose membrane (Invitrogen, Life Tech- 9 9 9 nologies). After saturation of nonspecific binding sites by incubating the TGCACC-3 and antisense, 5 -GCAAAGTTGACCTGACAGAC-3 . blot for 1 h in TBS containing 20 mM Tris-HCl, 5% nonfat dry milk, and DNA pull-down assay 0.1% Tween 20, the membrane was probed with specified primary Ab, followed by appropriate secondary HRP-conjugated Ab, and then revealed A DNA fragment containing ITGAE promoter or ITGAE enhancer was by chemoluminescence using SuperSignal WestPico substrate (Pierce/ amplified by PCR using a 59 biotin-labeled forward primer. Primer se- Perbio). quences were as follows: ITGAE promoter, forward, 59-ATAGAGCTCC- ATCGCCACTCTGCACTTCCAGCAGCC-39 and reverse, 59-ATACTC- Confocal microscopy GAGCATCCTTGCTGGAGCAGAGGCGGCTGTG-39;andforITGAE enhancer, forward, 59-ATAGGATCCCATTTACATGGGGTCTTGTTCTG- T cells were stimulated with a combination of anti-CD3 mAb (UCHT1) and 9 9 rTGF-b1 in the absence or presence of SB, SIS3, or CsA (100 nM), and TCACCCAGG-3 and reverse, 5 -ATAGTCGACCATGAGCAAGGATA- TAGAAGATATGAACAAC-39. The biotinylated probes (1 mg) were mixed then plated on poly(L-lysine)-coated coverslips (Sigma-Aldrich). Cells m were then fixed with 4% paraformaldehyde for 30 min and permeabilized with cell lysates in 400 l of a binding buffer (25 mM HEPES [pH 7.9], 50 mM NaCl, 1 mM MgCl2,1mMDTT,2mg polydI-dC) and incubated with 0.1% SDS or Triton X-100 for 10 min, followed by blocking with m 10% FBS for 20 min. The fixed cells were stained with anti-Smad2, anti- for 2 h at 4˚C. Then 25 l streptavidin–agarose beads (Invitrogen) were Smad3, or anti–NFAT-1 mAb and then with a secondary mAb coupled to added and incubated for an additional 1 h. The streptavidin–agarose beads Alexa-Fluor-488 (Molecular Probes, Invitrogen). All Ab were diluted in were washed five times with the binding buffer, and then SDS-sample PBS containing 1 mg/ml BSA. Nuclei were stained with TO-PRO-3 iodide buffer was added. The complexes were subjected to SDS-PAGE, followed (Molecular Probes, Invitrogen). Coverslips were mounted with Vectashield by immunoblotting with anti–phospho-Smad3 or anti–NFAT-1 mAb. (Vector laboratories) and analyzed the following day using a fluorescence Statistical analysis microscope (Carl Zeiss LSM-510). Z-projection of slices was performed using LSM Image Examiner software (Carl Zeiss). Nuclear translocation Data were compared using the two-tailed Student t test. Two groups were of transcription factors was monitored. considered as significantly different if p , 0.05. The Journal of Immunology 2473

Results CTL toward specific tumor cells (1, 2). Indeed, as shown in Fig. 2D, Smad2 and Smad3 transcription factors are activated after untreated H32-22 T cells, which do not express CD103 (Fig. 2A), TGF-b1 stimulation of CD8 T cells are unable to lyse the autologous lung cancer cell line IGR-Heu. In contrast, T lymphocytes expressing high levels of CD103, after Using a human cytotoxic T cell clone model, H32-22, generated TGF-b1 plus anti-CD3 mAb treatment, efficiently kill the specific from PBL of a lung cancer patient, we had previously demonstrated target. In concordance with CD103 expression analyses (Fig. 2A), the key role played by integrin CD103, induced upon stimulation we found that the TGF-bR1 inhibitor SB totally inhibited tumor with TGF-b1 and anti-CD3 mAb, in triggering specific TCR- cell lysis, whereas SIS3 had only a partial effect (Fig. 2D). Together mediated tumor cell lysis (1). As Smad2 and Smad3 are critical these results demonstrate the essential role of the Smad pathway in transcription factors in TGF-b1 signaling (37), we investigated CD103 induction and CTL effector function triggered by TGF-b1 their contribution to this biological effect using our T cell model plus TCR signaling. system. Smad2 and Smad3 transcriptional activity is governed by their rapid phosphorylation and translocation into the nucleus after NFAT-1 is involved in CD103 expression cell stimulation. Therefore, we analyzed these two processes in the Induction of CD103 on CD8 T lymphocytes requires, along with H32-22 T cell clone. Fig. 1A and Supplemental Fig. 1 show that exposure to TGF-b1, the engagement of TCR through recognition both Smad2 and Smad3 were rapidly phosphorylated after T cell of the peptide–MHC class I complex on target cells (2) or via an clone activation with TGF-b1 or a combination of TGF-b1and anti-CD3 mAb. As data from Fig. 2A showed that TGF-b1 alone anti-CD3 mAb. Moreover, phosphorylation of the two proteins is not sufficient to induce CD103 expression and that the signal was inhibited in the presence of the TGF-bR1 kinase inhibitor SB given by the anti-CD3 mAb was also required for integrin in- Downloaded from or, for Smad3, the Smad3-specific inhibitor SIS3 (Fig. 1A). duction, we assumed that another pathway in addition to the Smad Phosphorylated Smad2 and Smad3 form complexes with Smad4 pathway could be involved in regulating CD103 expression. Among and translocate into the nucleus, where they regulate target gene the various transcription factors triggered through the TCR–CD3 transcription (38, 39). Experiments were therefore also performed complex in T cells is NFAT-1, a key protein in the network of to analyze Smad2 and Smad3 locations in untreated T cells or in molecules that initiate T cell activation. Moreover, molecular an- T cells treated with a combination of TGF-b1 and anti-CD3 mAb. alyses of the human ITGAE gene (see below) indicated the pres- http://www.jimmunol.org/ Confocal microscopy analyses indicated that H32-22 T cells cul- ence of consensus NFAT binding sites in its regulatory regions. We tured in the presence of TGF-b1 plus anti-CD3 displayed nuclear therefore investigated the activation of NFAT-1 and its involvement accumulation of Smad2 and Smad3 proteins (Fig. 1B, upper in CD103 expression in H32-22 T lymphocytes. We first monitored panels). Indeed, 92% 6 6 and 76% 6 6 of stimulated cells, com- NFAT-1 nuclear localization induced by a combination of TGF-b1 pared with 5% 6 1 and 3% 6 4 of unstimulated cells, exhibited a and anti-CD3 mAb. Confocal microscopy analyses indicated that, nuclear localization of Smad2 and Smad3, respectively (Fig. 1B, compared with unstimulated cells, H32-22 T cells stimulated with lower panels). We then assessed the effect of SB and SIS3 inhibitors TGF-b1 plus anti-CD3 displayed nuclear translocation of NFAT-1 on Smad protein localization after T cell treatment with TGF-b1 (Fig. 3A, left panel). Indeed, although only 14% 6 7 of untreated b and anti-CD3 mAb. Results indicated that addition of the TGF- R1 cells displayed spontaneous nuclear localization of NFAT-1, by guest on September 24, 2021 kinase inhibitor SB resulted in total inhibition of nuclear translo- 93% 6 4 of T lymphocytes stimulated with TGF-b1 plus anti- cation of both transcription factors (Fig. 1B, upper panels). As CD3 showed accumulation of the transcription factor in the expected, SIS3 only abrogated Smad3 translocation into the nu- nucleus (Fig. 3A, right panel). cleus, whereas it had no effect on Smad2 localization. Indeed, We then assessed the effect of the calcineurin inhibitor CsA, only 2% 6 3 and 5% 6 2 of SB-treated cells displayed nuclear which inhibits NFAT-1 activation (40). Results indicate that CsA Smad2 and Smad3 expression, whereas 86% 6 20 and 1% 6 1of totally blocked NFAT-1 relocalization in the nucleus (Fig. 3A). SIS3-treated cells showed nuclear accumulation of Smad2 and CsA also led to a dramatic decrease in CD103 expression on H32- Smad3, respectively (Fig. 1B, lower panels). 22 and TALL-104 T cells induced by TGF-b1 plus anti-CD3 Smad3 cooperates with Smad2 in TGF-b1–induced CD103 treatment (Fig. 3B, Supplemental Fig. 2) and suppressed H32- expression on TCR-activated CD8 T cells and TCR-mediated 22–mediated cytotoxicity toward autologous IGR-Heu tumor cells cytotoxicity (see Fig. 2D). These data support a role for NFAT-1 in induction of CD103 on CD8 T lymphocytes upon TCR engagement. They also Next, we used SB and SIS3 inhibitors to analyze the involvement indicate that TGF-b1 and TCR signaling pathways cooperate to of Smad2 and Smad3 in CD103 induction at the T cell clone induce CD103 and to trigger the cytotoxic function of CD8+ CTL. surface. Unstimulated H32-22 T cells do not spontaneously express CD103. However, as we had previously reported (1), whereas TGF-b1 ITGAE gene regulatory regions include promoter and enhancer or anti-CD3 mAb used alone were unable to induce integrin ex- elements with Smad and NFAT binding sites pression on H32-22 CTL, their combination induced strong expres- ITGAE gene regulatory elements are still poorly defined. To iden- sion of CD103 (Fig. 2A). This increase was completely inhibited tify human ITGAE gene regulatory regions, we used Genomatix by SB and partially by SIS3. Similar results were obtained with the software that can predict gene promoter sequences and transcription CD8+ leukemia T cell line TALL-104, in which CD103 expression factor binding sites. Analyses of the whole sequence of the ITGAE induced by TGF-b1 and anti-CD3 was again strongly inhibited by gene (86.62 Kb) identified two potential regulatory regions with SB and to a lesser extent by SIS3 (Supplemental Fig. 2). Because consensus binding sites for both Smad and NFAT proteins, which Smad2-specific chemical inhibitors are not available, and to address may correspond to the ITGAE promoter and enhancer regions the role of this transcription factor in CD103 expression, we used a (Supplemental Fig. 3A). The potential promoter (regulatory se- specific siRNA that completely blocks Smad2 expression in H32-22 quence 1, see Supplemental Fig. 3B) starts upstream of exon 1 cells (Fig. 2B). Results shown in Fig. 2C indicate that Smad2 knock- and ends 32 bp before its 39-terminal end (2801 to +101). It con- down poorly affects CD103 induction by TGF-b1 plus anti-CD3 mAb. tains both a NFAT consensus binding site (59-TCTTTCCA-39)anda We had previously reported, at the functional level, the essential sequence (59-CTGAGATGTCTGG-39) encompassing a Smad role exerted by CD103 in triggering TCR-mediated cytotoxicity of binding site (59-GTCT-39) separated by 145 nucleic acids (Fig. 4A, 2474 ROLE OF Smad AND NFAT IN ITGAE GENE EXPRESSION

FIGURE 1. Activation of Smad2 and Smad3 after T cell clone stimulation with TGF-b1. (A) Western blot analysis of Smad2 and Smad3 protein phosphorylation following stimulation of the H32- 22 T cell clone, pretreated or not with SB or SIS3 inhibitor, with plastic-coated anti-CD3mAbandTGF-b1for1h. T cells stimulated with anti-CD3 mAb alone or TGF-b1 alone were included as controls. Actin was used as a loading Downloaded from control. Lower panels show normaliza- tion of phosphorylated Smad proteins relative to total Smad. Data shown represent one of three independent experiments. (B) Confocal microscopy analysis of nuclear relocalization of Smad2 and

Smad3 transcription factors. H32-22 http://www.jimmunol.org/ T cells, pretreated or not with TGF-bR1 kinase (SB) or Smad3 (SIS3) inhibitor, were stimulated for 1 h with a combination of anti-CD3 and rTGF-b1 and then stained with anti-Smad2 (left panel)or anti-Smad3 (right panel) mAb (green fluorescence). Nuclei were stained with TO-PRO-3 iodide (red fluorescence). Original magnification 363. Lower panels by guest on September 24, 2021 show percentages of T cells displaying Smad2 (left)orSmad3(right) nuclear relocalization. Data shown represent mean 6 SD of two independent experiments. Numbers of cells analyzed (n =50).

upper panel). The potential enhancer (regulatory sequence 2, see Binding of the two transcription factors to potential ITGAE gene Supplemental Fig. 3C) is located within intron 1 and extends from regulatory elements was further confirmed by the DNA pull-down position +16,561 to +17,462. It also includes a Smad binding site assay using nuclear extracts from H32-22 T cells stimulated with (59-AGAC-39) and a NFAT consensus binding site (59-TTTTTCCA- TGF-b1 and/or anti-CD3 mAb (Supplemental Fig. 4A). 39) separated by 27 nucleic acids (Fig. 4A, lower panel). Next, using the ChIP assay, we analyzed the binding capacity of The activity of ITGAE promoter and enhancer regions is Smad2, Smad3, and NFAT-1 to these DNA regulatory sequences dependent on Smad and NFAT transcription factors using ChIP assay-validated anti-Smad2, anti–phospho-Smad3, and To analyze the capacity of the sequences identified above to regulate anti–NFAT-1 Ab. An anti-IgG mAb was included as a control. transcription of the ITGAE gene, luciferase reporter constructs in Substantial binding of phospho-Smad3 to the Smad binding site of a vector with no promoter and containing either whole regulatory the potential ITGAE promoter was detected in immune complexes sequence 1 or this sequence together with the potential enhancer with DNA prepared from H32-22 T cells stimulated with TGF-b1 element were prepared and tested in 293-T cells untreated or treated plus anti-CD3 (Fig. 4B). In contrast, no amplification could be with TGF-b1. No luciferase activity was detected with regulatory seen with anti-Smad2 and anti–NFAT-1 immunoprecipitates. With sequence 1 (Fig. 5A). In contrast, luciferase activity was detected regard to the potential enhancer element, both phospho-Smad3 and in cells transfected with a reporter vector also containing the en- NFAT-1 bound to the regulatory sequence, but not Smad2 (Fig. 4C). hancer sequence (Supplemental Fig. 4B). Notably, deletion of Smad The Journal of Immunology 2475

FIGURE 2. (A) Role of Smad2 and Smad3 in CD103 induction on the T cell clone surface. CD1032 T cell clone H32- 22 was either untreated or pretreated with TGF-bR1 kinase (SB) or Smad3 (SIS3) inhibitor for 1 h and then stimulated with a combination of plastic-coated anti-CD3 mAb and rTGF-b1for4d.Tcells stimulated under the same conditions with anti-CD3 alone or rTGF-b1 alone were included as controls. Cells were then analyzed for aEb7 integrin expression by immunofluorescence staining using anti-CD103 (black fill) mAb. An isotypic control mAb was included (without fill). Percentages of positive Downloaded from cells are indicated. Numbers in parentheses correspond to mean fluorescence intensity of positive cells. (B) Western blot analysis of Smad2 protein following mRNA knockdown using specific siRNA-S2. Luciferase siRNA (siRNA- http://www.jimmunol.org/ Luc) was used as a negative control. Actin was used as a loading control. (C) Role of Smad2 in CD103 induction on the T cell clone surface. The H32- 22 T cell clone was electroporated with siRNA-S2 or siRNA-Luc control and stimulated with plastic-coated anti-CD3 plus TGFb-1. Cells were then analyzed by immunofluorescence labeling using anti-CD103 (black fill) or an isotypic by guest on September 24, 2021 control (without fill) mAb. (D) Role of CD103 in T clone–mediated lysis toward autologous tumor cells. T cell clone H32- 22, untreated or pretreated with SB, SIS3, or CsA, was unstimulated or stimulated with a combination of anti-CD3 mAb and TGF-b1, and then cytotoxic activity against the IGR-Heu tumor cell line was determined by a conventional 51Cr- release assay at indicated E:T ratios. Data shown represent one of three independent experiments.

binding sites in the promoter and enhancer core (Fig. 5B) or use of Jurkat-Tag T cells (Supplemental Fig. 4E). The constitutive phos- an antisense enhancer (Supplemental Fig. 4C) inhibited luciferase phorylation of Smad2 and Smad3 in this cell line (see Supplemental activity. It should be noted that stronger activity was not observed Fig. 4F) is most likely responsible, a hypothesis also supported by when cells were stimulated with TGF-b1 (Supplemental Fig. 4B, its inhibition in the presence of SB. Importantly, stimulation of Fig. 5B). These results are most likely due to the presence of Jurkat-Tag T cells with anti-CD3 mAb induced an increase in lu- constitutive active Smad molecules in 293-T cells, as suggested ciferase activity, further supporting a role of TCR signal in ITGAE by Western blot analysis revealing spontaneous phosphoryla- gene expression regulation (Supplemental Fig. 4E). tion of both proteins in this cell line (Supplemental Fig. 4D). We then assessed the contribution of NFAT-1 to ITGAE enhancer Therefore, and to conﬁrm their involvement in the observed ac- activity. With this aim, we analyzed the activity of a reporter vector tivity, cells were pretreated with the TGF-bR1 kinase inhibitor containing the two regulatory sequences in 293-T cells, displaying SB. Results show that cell treatment with SB totally abrogated constitutively phosphorylated Smad2 and Smad3 (see Supplemental luciferase activity (Fig. 5C). A spontaneous luciferase activity was Fig. 4D), cotransfected with a NFAT-1–encoding plasmid. No also obtained with the ITGAE reporter plasmid transfected in luciferase activity was observed in untreated cells. In contrast, 2476 ROLE OF Smad AND NFAT IN ITGAE GENE EXPRESSION

FIGURE 3. Role of NFAT-1 in CD103 induction on the T cell clone surface. (A) Confocal microscopy analysis of nuclear translocation of NFAT-1 transcription factor. H32-22 T cells, untreated or pretreated with CsA, were stimulated for 1 h with anti-CD3 plus TGFb-1 and then stained with anti–NFAT-1 mAb (green fluorescence). Nuclei were stained with TO-PRO-3 iodide (red fluorescence). Original magnification 363. Right panel, Percentages of T cells displaying NFAT-1 nuclear relocalization. Data shown represent mean 6 SD of two independent experiments. Numbers of cells analyzed

(n = 50). (B) Expression of the aEb7 integrin on the CD8+ T cell clone sur- Downloaded from face. H32-22 T cells, untreated or pretreated with CsA, were stimulated with a combination of plastic-coated anti-CD3 mAb and rTGFb-1 for 4 d and then analyzed by immunofluorescence staining using anti-CD103 (black fill) mAb. T cells stimulated with anti-CD3 alone or rTGF- http://www.jimmunol.org/ b1 alone and an isotypic control (without fill) mAb were included. Percentages of positive cells are indicated. Numbers in parentheses correspond to mean fluorescence intensity of positive cells. by guest on September 24, 2021 treatment of 293-T cells with PMA plus ionomycin, known to recruitment and phosphorylation of receptor-associated Smad2 and strongly activate NFAT-1, triggered an increase in luciferase ac- Smad3 molecules (37). Upon their phosphorylation, Smad2 and tivity (Fig. 5D). Collectively, these results indicate that Smad3 Smad3 undergo homotrimerization and formation of hetero- effectively binds to the proximal regulatory sequence and that oligomeric complexes with the co-Smad molecule, Smad4. These both Smad3 and NFAT-1 bind to the distal regulatory region. They activated Smad complexes enter the nucleus and, in cooperation also support the hypothesis that these DNA sequences truly cor- with other transcription factors, regulate transcription of target respond to ITGAE gene promoter and enhancer, respectively. genes (37). Our data indicate that T cell treatment with TGF-b1 induces Smad2 and Smad3 phosphorylation and their nuclear Discussion translocation. They also indicate that CD103 induction at the TGF-b1 is involved in CD103 induction upon TCR engagement surface of CD8+ T cells requires activation of these transcription (31, 32). Indeed, we show in this work that both signaling path- factors. Indeed, inhibition of TGF-bR1 abrogated CD103 induc- ways are required for CD103 induction on CD8 T cell surface. tion by TGF-b1 and, concomitantly, specific TCR-mediated tumor TGF-b1 is frequently described as an immunosuppressive cyto- cell lysis. In contrast, inhibition of either Smad3 or Smad2 sepa- kine used by cancer cells to escape from the immune system and rately had only a weak or marginal effect, suggesting that a Smad- target CTL functions (41). Therefore, the paradoxical role of TGF-b1 independent pathway is involved in CD103 expression regulation, in optimization of antitumor CTL reactivity via regulation of the or, alternatively, that Smad2 and Smad3 are redundant for TGF-b– CD103 integrin is an important observation that warrants intensive mediated induction of CD103. This result is intriguing because, in investigation. In this report, using a human tumor Ag-specific CTL principle, Smad3 binds to DNA, whereas Smad2 does not (38), and system model, we demonstrated that Smad2/3 transcription fac- we did not find any binding of Smad2 to ITGAE promoter or en- tors, in cooperation with NFAT-1, are directly involved in CD103 hancer elements. Thus, it remains unclear as to how Smad2 upre- induction on CD8+ T cells upon TCR engagement in the presence gulates expression of ITGAE when Smad3 is inhibited. One of TGF-b1. We also identified both promoter and enhancer reg- possibility is that Smad2 activates transcription of the gene by ulatory sequences of the human CD103-encoding ITGAE gene interacting with other yet unknown transcription factors, whose involved in this process. binding regions are distinct from the Smad3 binding sites identi- TGF-b1 initiates signaling by binding to TGF-bR at the cell mem- fied in the present report. Further studies will be necessary to brane to form a heterotetrameric complex composed of TGF-bR clarify this issue, but a recent report showing that Smad2 and type I and type II dimers. These receptors are serine/threonine Smad3 are redundantly essential for TGF-b–mediated regulation kinase receptors, and, in this complex, TGF-bR2 catalyzes phos- of Treg cell plasticity and Th1 development reached the same phorylation of the cytoplasmic domain of TGF-bR1. This leads to conclusion (42). CD103 induction on CD8 T cells also requires an The Journal of Immunology 2477

FIGURE 4. (A) Schematic localization of Smad and NFAT binding sites on the ITGAE gene promoter and enhancer regions. Potential NFAT and Smad binding sequences are represented in bold italics and separated by indicated base pair numbers. Arrows indicate oligonucleotide probes used for ChIP experiments. (B) ChIP analysis of Smad- and NFAT-1–binding capacity to the ITGAE promoter region. H32-22 T cells were either kept unstimulated or stimulated with anti-CD3 mAb plus TGFb-1, and cell lysates were immunoprecipitated with either anti-Smad2, anti–phospho-Smad3 Downloaded from (left panel), anti–NFAT-1, or anti-IgG negative control (right panel). Immu- noprecipitates were then subjected to quantitative RT-PCR using ITGAE promoter primers encompassing Smad or NFAT sites. Values are normalized to input genomic DNA. (C) ChIP analysis http://www.jimmunol.org/ of Smad- and NFAT-1–binding capacity to the ITGAE enhancer region. H32-22 T cells were either kept unstimulated or stimulated with a combination of anti- CD3 and TGFb-1, and cell lysates were then immunoprecipitated with anti- Smad2, anti–phospho-Smad3 (left panel), anti–NFAT-1, or anti-IgG negative control

(right panel). Immunoprecipitates were by guest on September 24, 2021 analyzed by quantitative RT-PCR using ITGAE enhancer-speciﬁc primers encompassing Smad and NFAT sites. Values are normalized to input genomic DNA.

additional signal given by the TCR. Our data point to the im- most likely because these cells constitutively express phosphory- portant role played by NFAT-1 in this function, as illustrated by lated Smad2 and Smad3. Indeed, it was abrogated with the TGF- the dramatic blocking effect of the calcineurin inhibitor CsA upon bR1 inhibitor or after deletion of the Smad binding site in the CD103 expression on T lymphocytes treated with TGF-b1 plus regulatory sequence. Taken together, these data show that tran- anti-CD3 and also the complete inhibition of CTL-mediated lysis scription regulation of the ITGAE gene is more complex than that of autologous tumor cells. Collectively, these results show that of other integrin genes, such as ITGAL, ITGA2, ITGA6,andITGAX Smad and NFAT transcription factors cooperate to induce CD103 (43–47), for which promotor regions directed tissue-specific func- expression and thus trigger the CTL effector function. tional activity, and that ITGAE expression is regulated through In the current study, we also identified an ITGAE promoter promoter and enhancer elements with the participation of both located upstream of exon 1, which roughly corresponds to that Smad and NFAT-1 transcription factors. It is to note that, as op- previously suggested by Robinson et al. (34). We also identified an posed to CD103 integrin, we observed that TGF-b1 had an in- ITGAE enhancer located within intron 1. Each regulatory region hibitory effect on LFA-1 (aLb2) expression levels on the surface includes Smad and NFAT consensus binding sites, but whereas of the T cell clone used in the current study (data not shown). both ITGAE regulatory sequences effectively bind Smad3, only We previously reported, in a xenograph model, that stable ex- the enhancer region is able to bind NFAT-1. In agreement with a pression of CD103 can be induced in vivo following H32-22 T cell previous report (34), we found that the ITGAE promoter sequence transfer into the autologous tumor and interaction of TCR with the by itself, without any enhancer, is inactive in a reporter gene ac- specific tumor epitope–MHC-I complex presented on the surface tivation assay in 293-T cells. However, enhancer activity was of malignant cells. We also reported that neutralization of TGF-b1, detected with a reporter plasmid containing both ITGAE promoter secreted within the tumor microenvironment with a soluble AdTGF- and enhancer elements. This activity was constitutive in 293-T cells, b1RII-Fc molecule, inhibited ITGAE expression in tumor-reactive 2478 ROLE OF Smad AND NFAT IN ITGAE GENE EXPRESSION Downloaded from http://www.jimmunol.org/

FIGURE 5. Role of Smad3 and NFAT-1 in ITGAE promoter and enhancer activities. (A) Luciferase assay of the ITGAE reporter plasmid. Promoter activity was analyzed in 293-T cells, either kept unstimulated (medium) or stimulated with TGFb-1. An empty vector negative control was included. Bars correspond to the mean of relative light units (RLU) 6 SD of two independent experiments from four. (B) Enhancer activity of the luciferase reporter plasmid containing ITGAE promoter and enhancer. The 293-T cells were transfected with the reporter plasmid containing both ITGAE promoter and enhancer sequences, either nonmutated or mutated for Smad binding sites, and then cultured in medium or in the presence of TGFb-1. (C) Role of Smad in ITGAE enhancer activity. The 293-T cells, pretreated with the TGF-bR1 kinase inhibitor SB, were transfected with the reporter plasmid containing both the ITGAE promoter and enhancer and then cultured in medium or in the presence of TGFb-1. (D)RoleofNFAT-1inITGAE enhancer activity. by guest on September 24, 2021 Luciferase assay was performed in 293-T cells transfected with the reporter plasmid containing both the ITGAE promoter and enhancer and then transduced with a NFAT-1 plasmid in the absence or presence of PMA plus ionomycin to induce translocation of the transcription factor into the nucleus. *p , 0.05, **p , 0.01.

CD8+ T cells (2). These results are fully consistent with the Treg cell subpopulation that accumulates within tumors and favors conclusion that Smad and NFAT pathways are key regulators in cancer cell escape from immune responses. initiating transcription of the ITGAE gene. However, additional In summary, we have identified an ITGAE gene enhancer ele- transcription factors, such as Runx, may also be involved in ITGAE ment and defined the role of Smad and NFAT-1 transcription gene regulation under in vivo conditions. Indeed, evidence for in- factors in CD103 induction. These results support the conclusion volvement of Runx factors in the TGF-b pathway and interactions that TGF-b1 plays a major role in activation of CD103 in CD8 between Smad and Runx proteins have been previously reported T cells and that this cytokine contributes to regulating antitumor (48–51). Runx binding regions were also found in the ITGAE immune responses. Our studies also suggest a rational immuno- regulatory sequences identified in this work (data not shown), and therapeutic approach for inducing CD103+ T cells by specific Runx3 has been shown to induce CD103 expression during de- activation of signals that increase Smad-dependent ITGAE gene velopment of CD42/CD8+ T cells or on a subpopulation of CD4+/ expression in tumor-specific TCR-activated T cells. These will not CD82 T cells having Treg cell characteristics (52). only increase the capacity of TGF-b1 to enhance CD103 ex- Regulation of CD103 expression appears similar to that of Foxp3 pression on tumor-infiltrating T lymphocytes and their killing in several aspects, as follows: 1) both proteins are expressed fol- activity but will also improve their retention at the tumor site. lowing T lymphocyte activation by a combination of TGF-b1 and anti-CD3; 2) ITGAE and Foxp3 genes are regulated by enhancer elements, including both Smad3 and NFAT binding sites; and 3) Acknowledgments they are induced by enhancers through the actions of Smad3 and We thank Y. Lecluse for FACS analyses, S. Salome´-Desmoulez for confo- NFAT (53). Interestingly, a subset of Treg cells coexpresses both cal microscopy, and I. Vergnon for T cell clone culture. We also thank C. Pouponnot, J. Raingeaud, and U. Maurer for Smad and NFAT constructs; Foxp3 and CD103 molecules (54). These Treg cells might be A. Atfi, M. Mangeney, F. Djenidi, M. Khaled, and A. Le Floc’h for helpful generated through interaction of TCR with tumor Ag, and, in this discussion; and N. Theret for critical reading of the manuscript. case, CD103 may control their retention at the tumor site (55), as suggested by studies in epithelial tissues (56), including epithelial tumors (2). However, the effective role of CD103 in Treg cell Disclosures functions is to date unknown, especially in the immunosuppressive The authors have no financial conflicts of interest. The Journal of Immunology 2479

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