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Leukocyte Function-associated Antigen-1/Intercellular Adhesion Molecule-1 Interaction Induces a Novel Genetic Signature Resulting in T-cells Refractory to Transforming Growth Factor-β Signaling*

  • Navin K. Verma
    Footnotes
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Eugene Dempsey
    Footnotes
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Aideen Long
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Anthony Davies
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Sean P. Barry
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Padraic G. Fallon
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland

    National Children's Research Centre, Our Lady's Children's Hospital, Dublin 12, Ireland
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  • Yuri Volkov
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Dermot Kelleher
    Correspondence
    To whom correspondence should be addressed: Inst. of Molecular Medicine, Trinity Centre for Health Sciences, Dublin 8, Ireland. Tel.: 353-1-896-1476; Fax: 353-1-454-2043
    Affiliations
    Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland
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  • Author Footnotes
    * This work was supported by grants from the Enterprise Ireland, Higher Education Authority of Ireland under the Program for Research in Third Level Institutions (PRTLI) Cycle 3, Science Foundation Ireland (SFI), and the Health Research Board (HRB) of Ireland.
    This article contains supplemental Table 1.
    1 Both authors contributed equally to this work.
Open AccessPublished:June 15, 2012DOI:https://doi.org/10.1074/jbc.M112.376616
      The immunesuppressive cytokine TGF-β plays crucial regulatory roles in the induction and maintenance of immunologic tolerance and prevention of immunopathologies. However, it remains unclear how circulating T-cells can escape from the quiescent state maintained by TGF-β. Here, we report that the T-cell integrin leukocyte function-associated antigen-1 (LFA-1) interaction with its ligand intercellular adhesion molecule-1 (ICAM-1) induces a genetic signature associated with reduced TGF-β responsiveness via up-regulation of SKI, E3 ubiquitin-protein ligase SMURF2, and SMAD7 (mothers against decapentaplegic homolog 7) genes and proteins. We confirmed that the expression of these TGF-β inhibitory molecules was dependent on STAT3 and/or JNK activation. Increased expression of SMAD7 and SMURF2 in LFA-1/ICAM-1 cross-linked T-cells resulted in impaired TGF-β-mediated phosphorylation of SMAD2 and suppression of IL-2 secretion. Expression of SKI caused resistance to TGF-β-mediated suppression of IL-2, but SMAD2 phosphorylation was unaffected. Blocking LFA-1 by neutralizing antibody or specific knockdown of TGF-β inhibitory molecules by siRNA substantially restored LFA-1/ICAM-1-mediated alteration in TGF-β signaling. LFA-1/ICAM-1-stimulated human and mouse T-cells were refractory to TGF-β-mediated induction of FOXP3+ (forkhead box P3) and RORγt+ (retinoic acid-related orphan nuclear receptor γt) Th17 differentiation. These mechanistic data suggest an important role for LFA-1/ICAM-1 interactions in immunoregulation concurrent with lymphocyte migration that may have implications at the level of local inflammatory response and for anti-LFA-1-based therapies.

      Introduction

      The multistep process of lymphocyte transmigration is crucial for an efficient immune response, and it is orchestrated by a multifunctional molecular array including integrin-mediated adhesions and chemokine signals (
      • Ley K.
      • Laudanna C.
      • Cybulsky M.I.
      • Nourshargh S.
      Getting to the site of inflammation: The leukocyte adhesion cascade updated.
      ). The leukocyte function-associated antigen-1 (LFA-1)
      The abbreviations used are: LFA-1
      leukocyte function-associated antigen-1
      ICAM-1
      intercellular adhesion molecule-1
      AP-1
      activator protein-1
      iTreg
      induced regulatory T-cell
      PBL
      peripheral blood lymphocyte
      qRT-PCR
      quantitative RT-PCR
      eGFP
      enhanced GFP.
      integrin interacts with its ligand intercellular adhesion molecule-1 (ICAM-1), expressed on the surface of high endothelial venules and other cell types, and this interaction plays key signaling roles in the context of leukocyte adhesion, locomotion, and migration through endothelial junctions into the sites of inflammation (
      • Hogg N.
      • Laschinger M.
      • Giles K.
      • McDowall A.
      T-cell integrins: More than just sticking points.
      ). At the level of the immunological synapse, LFA-1 greatly increases the avidity of T-cells and antigen presenting cells (
      • Hogg N.
      • Laschinger M.
      • Giles K.
      • McDowall A.
      T-cell integrins: More than just sticking points.
      ,
      • Shimizu Y.
      LFA-1: More than just T cell velcro.
      ). At the point of transendothelial migration, lymphocytes are directed toward an ultimate effector phase of their expected activity at the site of inflammation downstream of the high endothelial venules. In this regard, LFA-1/ICAM-1 interactions can modulate the signal transduction pathways that control complex cell functions such as T-cell activation and differentiation, which require the regulation of gene expression.
      We have previously shown that LFA-1 engagement in T-cells activates the transcription factor and adaptor protein STAT3 (
      • Verma N.K.
      • Dourlat J.
      • Davies A.M.
      • Long A.
      • Liu W.Q.
      • Garbay C.
      • Kelleher D.
      • Volkov Y.
      STAT3-stathmin interactions control microtubule dynamics in migrating T-cells.
      ), whereas others have shown that such engagement activates c-Jun that forms the activator protein-1 (AP-1) transcriptional regulatory complex (
      • Shimizu Y.
      LFA-1: More than just T cell velcro.
      ,
      • Perez O.D.
      • Mitchell D.
      • Jager G.C.
      • South S.
      • Murriel C.
      • McBride J.
      • Herzenberg L.A.
      • Kinoshita S.
      • Nolan G.P.
      Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1.
      ,
      • Bianchi E.
      • Denti S.
      • Granata A.
      • Bossi G.
      • Geginat J.
      • Villa A.
      • Rogge L.
      • Pardi R.
      Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity.
      ). In addition, STAT3 interacts with c-Jun and participates in cooperative transcriptional activation (
      • Zhang X.
      • Wrzeszczynska M.H.
      • Horvath C.M.
      • Darnell Jr., J.E.
      Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation.
      ). These observations suggest that signaling through LFA-1 may affect downstream gene expression. Although extensive progress has been made in understanding the LFA-1 signaling at a post-translational level, detailed characterization of gene regulation by LFA-1/ICAM-1 interaction and its functional consequences in T-cells has not been explored to the similar extent. Moreover, the precise contribution of the LFA-1/ICAM-1 signaling to the enhanced extravasation capacity of effector T-cells at sites of inflammation has remained unclear.
      A balance between pro- and anti-inflammatory mechanisms at the epithelial interfaces allows for efficient protection against pathogens yet preventing adverse inflammation. Central to the success of immune responses that restrain inflammation are regulatory cytokines, including a multifunctional cytokine TGF-β produced by immune and non-immune cells that controls a number of immunological functions, including T-cell development, homeostasis, proliferation, and differentiation (
      • Rubtsov Y.P.
      • Rudensky A.Y.
      TGFβ signaling in control of T-cell-mediated self-reactivity.
      ,
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ,
      • Li M.O.
      • Sanjabi S.
      • Flavell R.A.
      Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
      ). TGF-β signaling from the cell surface to the nucleus is mediated by the SMAD family of proteins that involves Ser/Thr phosphorylation of receptor-regulated SMAD2 and SMAD3 (
      • Moustakas A.
      • Souchelnytskyi S.
      • Heldin C.H.
      Smad regulation in TGF-β signal transduction.
      ). This process is subject to many levels of positive and negative regulation by intracellular mediators. Among negative regulators of TGF-β signaling are SMAD7, SMURF2, SKI, and SnoN (
      • Moustakas A.
      • Souchelnytskyi S.
      • Heldin C.H.
      Smad regulation in TGF-β signal transduction.
      ,
      • Nakao A.
      • Afrakhte M.
      • Morén A.
      • Nakayama T.
      • Christian J.L.
      • Heuchel R.
      • Itoh S.
      • Kawabata M.
      • Heldin N.E.
      • Heldin C.H.
      • ten Dijke P.
      Identification of Smad7, a TGFβ-inducible antagonist of TGF- β signaling.
      ,
      • Deheuninck J.
      • Luo K.
      Ski and SnoN, potent negative regulators of TGF-β signaling.
      ). The pleiotropic effects of TGF-β on various T-cell subsets are complex and context-dependent. TGF-β signaling prevents production of IL-2 by T-cells (
      • Das L.
      • Levine A.D.
      TGF-β inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction.
      ). Furthermore, TGF-β is a critical co-stimulatory factor in the differentiation of functional subsets of effector T helper 17 (Th17) cells as well as induced regulatory T-cells (iTregs) (
      • Rubtsov Y.P.
      • Rudensky A.Y.
      TGFβ signaling in control of T-cell-mediated self-reactivity.
      ,
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ,
      • Li M.O.
      • Sanjabi S.
      • Flavell R.A.
      Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
      ), which play pivotal roles in the control of immune homeostasis. Th17 cells are characterized by their expression of retinoic acid-related orphan receptor (RORγt) and production of proinflammatory cytokines, including IL-17, IL-6, and IL-21 (
      • Weaver C.T.
      • Hatton R.D.
      Interplay between the TH17 and TReg cell lineages: A (co-)evolutionary perspective.
      ). Th17 cells reside mainly at barrier surfaces, particularly the mucosa of the gut, where they function to protect the host from microorganisms that invade through the epithelium (
      • Weaver C.T.
      • Hatton R.D.
      Interplay between the TH17 and TReg cell lineages: A (co-)evolutionary perspective.
      ). The iTreg cells, which develop post-thymically are characterized by expression of the transcription factor FOXP3 (forkhead box protein P3), prevent tissue-specific autoimmunity and chronic inflammation (
      • Weaver C.T.
      • Hatton R.D.
      Interplay between the TH17 and TReg cell lineages: A (co-)evolutionary perspective.
      ,
      • Josefowicz S.Z.
      • Niec R.E.
      • Kim H.Y.
      • Treuting P.
      • Chinen T.
      • Zheng Y.
      • Umetsu D.T.
      • Rudensky A.Y.
      Extrathymically generated regulatory T cells control mucosal TH2 inflammation.
      ). The fact that the immunosuppressive cytokine TGF-β is expressed highly in lymphoid and extralymphoid organs and that constitutively active TGF-β signaling keeps circulating T-cells in a resting state brings about a fundamental question. How do circulating T-cells escape from the quiescent state maintained by TGF-β? In particular, it remains to be established whether LFA-1/ICAM-1-mediated signaling in T-cells has a functional involvement in these processes. Recent data from trials of monoclonal antibodies directed against LFA-1 in humans, as well as data from animal models, suggest that such therapies are associated not only with inhibition of migration but also with broad perturbations in T-cell functions, including activation, proliferation, and differentiation (
      • Koszik F.
      • Stary G.
      • Selenko-Gebauer N.
      • Stingl G.
      Efalizumab modulates T cell function both in vivo and in vitro.
      ,
      • Reisman N.M.
      • Floyd T.L.
      • Wagener M.E.
      • Kirk A.D.
      • Larsen C.P.
      • Ford M.L.
      LFA-1 blockade induces effector and regulatory T-cell enrichment in lymph nodes and synergizes with CTLA-4Ig to inhibit effector function.
      ).
      The purpose of the current study was to determine whether there are changes in gene expression caused by LFA-1/ICAM-1 interactions, which could have an impact not only on the T-cell locomotory behavior but also in functional terms on the programming of the lymphocyte diapedesis through the high endothelial venules and ultimate effector function. To address these questions, we analyzed gene expression changes following the interaction of LFA-1 with ICAM-1 in T-cells using an Affymetrix microarray technique and a systems biology approach. We demonstrate that among the genes whose expression levels are altered predictably by LFA-1/ICAM-1 mediated signaling, several may have functions related to the turnover of molecules involved in the migration process. Additionally, however, a subset of genes associated with reduced TGF-β responsiveness is up-regulated which may have an impact on the overall T-cell effector programming. In this study, we specifically address the impact of such gene expression regulation by LFA-1/ICAM-1 interactions in T-cells, which results in their refractoriness to TGF-β in terms of effector functions.

      EXPERIMENTAL PROCEDURES

      Cells and Reagents

      Human peripheral blood lymphocyte (PBL) T-cells from healthy volunteers purified by standard methods and the human T-cell line Hut78 (ATCC, Rockville, MI) were used. Human and mouse recombinant ICAM-1 and anti-human RORγt-APC antibody were from R&D Systems. LFA-1 blocking antibody, anti-CD4-FITC, and anti-FOXP3-PE were from BioLegend. Goat anti-human IgG (Fc-specific) and anti-α-tubulin antibodies were from Sigma. Rabbit anti-β-actin, HRP-conjugated anti-rabbit, and anti-mouse antibodies were from Cell Signaling Technology. Rabbit anti-SMAD7, anti-SMURF2, and anti-SKI were from Santa Cruz Biotechnology. Cell-permeable AG490, STAT3-specific inhibitor peptide, and JNK inhibitor II SP600125 were from Merck Millipore.

      LFA-1 Cross-linking with ICAM-1

      T-cell LFA-1 cross-linking to the immobilized ICAM-1 was performed using our migration-triggering model system as described (
      • Volkov Y.
      • Long A.
      • McGrath S.
      • Ni Eidhin D.
      • Kelleher D.
      Crucial importance of PKC-β(I) in LFA-1-mediated locomotion of activated T cells.
      ,
      • Volkov Y.
      • Long A.
      • Kelleher D.
      Inside the crawling T cell: Leukocyte function-asociated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isozymes β(I) and δ.
      ). This well defined system allows LFA-1/ICAM-1 interactions to be examined in the absence of any other receptor-ligand interactions. Briefly, tissue culture plates (flat bottom, NuncTM) were pre-coated with goat anti-human Fc-specific IgG and subsequently with human recombinant ICAM-1 (1 μg/ml). Control plates were coated with 0.01% poly-l-lysine or 1% bovine serum albumin. Cells were loaded into the coated wells (60 × 104 cells/well in a six-well plate) and incubated in 5% CO2 at 37 °C.

      Total RNA Extraction, DNA Microarray, and Data Analysis

      Total RNA was isolated from the T-cell samples using a Nucleospin II kit (Macherey-Nagel) as per the manufacturer's instructions. For each sample, 10 μg of total RNA was in vitro-transcribed and biotin-labeled using the GeneChip 3′-IVT express kit (Affymetrix). Quality of cRNA was assessed using a 2100 Bioanalyser in combination with the RNA nano chips (Agilent Technologies). Biotin-labeled fragmented cRNA was hybridized to U133 plus arrays (version 2.0) for 16 h. The chips were then washed, stained, and scanned. Affymetrix arrays were run in duplicate. Data analysis was performed in GeneSpring GX (version 7.3.1; Agilent Technologies). CEL files containing the raw signal intensities for each probe set were imported into Genespring and were normalized using GeneChip Robust Multiarray Averaging. Genes were selected on the criteria that they had changed by >1.5 fold with a significant p value (<0.05) after multiple correction testing using Benjamin and Hochberg FDR test.

      In Silico Analysis

      Ingenuity Pathways Analysis (IPA) (Ingenuity Systems) was performed to better understand experimental data in relation to published research by identifying relationships, functions, and pathways of relevance. To generate biological networks, the final list of differentially expressed genes was uploaded into the IPA software as a tab-delimited text file of gene IDs. The network is displayed as nodes that represent genes and edges representing the interactions between genes. The “IPA Path Designer” was used to generate the final network. The transcription factor binding sites in the promoters of the identified genes was identified using Text Mining Application and UCSC Genome Browser from SABiosciences (
      • Kent W.J.
      • Sugnet C.W.
      • Furey T.S.
      • Roskin K.M.
      • Pringle T.H.
      • Zahler A.M.
      • Haussler D.
      The human genome browser at UCSC.
      ).

      Quantitative Real-time PCR

      DiRE (
      • Gotea V.
      • Ovcharenko I.
      DiRE: Identifying distant regulatory elements of co-expressed genes.
      ) tool was used for promoter analysis and cDNA was generated using RETROscript qRT-PCR kit (Ambion). Real-time PCR was performed using 4.5 μl of diluted (1/50) reverse transcription reaction, TaqMan Universal PCR no AmpErase UNG master-mix, and specific gene primer set in a final volume of 10 μl in an ABI Prism 7700 thermocycler (Applied Biosystems). Relative quantification was performed using GAPDH as an internal control. Fold changes for each gene were calculated using the ΔΔCT method (
      • Heid C.A.
      • Stevens J.
      • Livak K.J.
      • Williams P.M.
      Real time quantitative PCR.
      ).

      Cell Lysis and Western Immunoblotting

      The cell lysis was performed as described previously (
      • Verma N.K.
      • Dempsey E.
      • Conroy J.
      • Olwell P.
      • Mcelligott A.M.
      • Davies A.M.
      • Kelleher D.
      • Butini S.
      • Campiani G.
      • Williams D.C.
      • Zisterer D.M.
      • Lawler M.
      • Volkov Y.
      A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin.
      ). The protein content of the cell lysates was determined by Bradford assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the cellular lysates and subsequent Western immunoblotting were performed as described (
      • Verma N.K.
      • Dempsey E.
      • Conroy J.
      • Olwell P.
      • Mcelligott A.M.
      • Davies A.M.
      • Kelleher D.
      • Butini S.
      • Campiani G.
      • Williams D.C.
      • Zisterer D.M.
      • Lawler M.
      • Volkov Y.
      A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin.
      ). Densitometric analyses of the Western blots were performed by using GeneTools software (Syngene). The relative values of the samples were determined by giving an arbitrary value of 1.0 to the respective control samples of each experiment (
      • Verma N.K.
      • Dempsey E.
      • Freeley M.
      • Botting C.H.
      • Long A.
      • Kelleher D.
      • Volkov Y.
      Analysis of dynamic tyrosine phosphoproteome in LFA-1 triggered migrating T-cells.
      ).

      Electroporation of T-cells

      Hut78 T-cells were electroporated using BTX ECM830 electroporator as per our previously optimized protocol (
      • Verma N.K.
      • Davies A.M.
      • Long A.
      • Kelleher D.
      • Volkov Y.
      STAT3 knockdown by siRNA induces apoptosis in human cutaneous T-cell lymphoma line Hut78 via down-regulation of Bcl-xl.
      ). Gene knockdown studies for the selected genes (human SMAD7, SMURF2, and SKI) were performed using SMARTpool® siRNA reagents (Dharmacon). For SMAD7 overexpression, cells were electroporated with an empty plasmid vector pcDNA3 or a construct FLAG-SMAD7 (a kind gift by professor Carl-Henrik Heldinm, Ludwig Institute for Cancer Research Ltd., Uppsala University, Uppsala, Sweden).

      Human T-cell Differentiation and Functional Assay

      Conversion of iTregs was performed as described (
      • Tran D.Q.
      • Ramsey H.
      • Shevach E.M.
      Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-β-dependent but does not confer a regulatory phenotype.
      ) with minor modifications. Briefly, PBL T-cells were stimulated with anti-human CD3/CD28-coated beads at a bead-to-cell ratio of 1:5 in the presence of 20 ng/ml IL-2 ± 5 ng/ml TGF-β (both from Peprotech) for 5 days. For RORγt+ Th17 differentiation, PBL T-cells were stimulated with anti-CD3/CD28 in the presence of 40 ng/ml IL-6 (Peprotech) ± 5 ng/ml TGF-β for 4 days. For blocking IL-2 in Th17 cultures, anti-IL-2, anti-CD122, and anti-CD25 antibodies were added (10 ng/ml each). Anti-IFN-γ and anti-IL-4 antibodies were also added (10 ng/ml each) to block Th1 and Th2 differentiation, respectively. CD4+ cells expressing FOXP3 or RORγt were detected by corresponding immunostaining and a cell-based automated microscopy (IN Cell Analyzer 1000, GE Healthcare). The percentage of CD4+ cells expressing FOXP3 or RORγt was quantified using IN Cell Investigator software (GE Healthcare).

      Mouse iTreg Differentiation and Analysis

      Ex vivo induction of iTreg in mouse T-cells was performed using Foxp3-eGFP reporter mice on a C57BL/6J background (The Jackson Laboratory). All animal experiments were performed in compliance with Irish Department of Health and Children regulations and approved by the Trinity College Dublin's BioResources Ethical Review Board. A single cell suspension was prepared from spleens as described (
      • Amu S.
      • Saunders S.P.
      • Kronenberg M.
      • Mangan N.E.
      • Atzberger A.
      • Fallon P.G.
      Regulatory B cells prevent and reverse allergic airway inflammation via FoxP3-positive T regulatory cells in a murine model.
      ). CD4+ T-cells were purified using the CD4+ T-cell isolation kit (Miltenyi) and plated in 48-well plates at a concentration of 1.0 × 106 cells/ml with or without 3 μg/ml plate bound mouse recombinant ICAM-Fc. For induction of iTregs, cells were stimulated for 72 h with 1.0 μg/ml plate-bound anti-CD3, 5.0 μg/ml soluble anti-CD28, and 20 ng/ml IL-2 in the presence or absence of 5 ng/ml TGF-β1. Cells were stained with anti-CD4 Peridinin Chlorophyll Protein Comlex-conjugated mAb (BD Biosciences) and surface marker expression of eGFP-positive cells was assessed by the CyAn Flow Cytometer (Beckman Coulter) using FlowJo software (TreeStar). Dead cells were excluded on the basis of propidium iodide staining.

      Cytokine Analysis

      Secreted levels of IL-2 and IL-17 in T-cell culture supernatants were measured by human IL-2 DuoSet® ELISA (R&D Systems) and LEGEND MAXTM human IL-17A ELISA Kit (BioLegends) as per the manufacturer's instructions.

      Statistical Analysis

      The data are expressed as mean ± S.E. For comparison of two groups, p values were calculated by two-tailed unpaired student's t test. In all cases, p values < 0.05 was considered to be statistically significant.

      RESULTS

      Analysis of Gene Regulation by LFA-1/ICAM-1-mediated Signaling in T-cells

      We compared the effect of LFA-1/ICAM-1 triggering in T-cells by recording changes in transcription profiles by microarray analysis. Human T-cells were stimulated on the immobilized ICAM-1 for 1, 3, or 6 h, and total RNA was extracted. RNA samples from five independent biological replicates were analyzed by GeneChip Human Genome U133Plus2.0 Array (Affymetrix) with genome-wide coverage using ∼54,000 probe sets (>47,000 transcripts). Data analysis as described in “Experimental Procedures” was performed to identify differentially expressed genes, treatment clusters, and affected biological pathways. There were significant changes in the expression profile of a range of genes induced through the LFA-1/ICAM-1-mediated signal. We identified a total of 195 genes, of which 144 were significantly up-regulated, and 51 genes were repressed by the LFA-1 signal. The full list of differentially expressed genes with details on their molecular function is contained in supplemental Table 1. A list of significantly up- and down-regulated genes (top 40 genes in each category) is provided in Table 1 and Table 2, respectively.
      TABLE 1The top 40 genes ranked by fold change identified as being up-regulated in T-cells following LFA-1/ICAM-1 interaction
      Probe IDSymbolGene nameFold change
      44783_s_atHEY1Hairy/enhancer-of-split related with YRPW motif 156.82
      222450_atTMEPAITransmembrane, prostate, androgen-induced RNA48.92
      217591_atSKILSKI-like15.48
      240156_atRFX2Regulatory factor X, 213.84
      235497_atLOC643837Hypothetical protein LOC64383713.55
      229613_atNKD1Naked cuticle 1 homolog (Drosophila)13.4
      213506_atF2RL1Coagulation factor II (thrombin) receptor-like 112.52
      230820_atSMURF2SMAD-specific E3 ubiquitin protein ligase 29.832
      209574_s_atC18orf1Chromosome 18 open reading frame 19.73
      1560676_atSIAH3Seven in absentia homolog 39.293
      1556423_atVASH1Vasohibin 19.273
      212907_atSLC30A1Solute carrier family 30 (zinc transporter), member 19.234
      216297_atBBIP1BBSome interacting protein 17.938
      218793_s_atSCML1Sex comb on mid leg-like protein 17.832
      232081_atABCG1ATP-binding cassette, sub-family G (WHITE), member 17.69
      212666_atSMURF1SMAD-specific E3 ubiquitin protein ligase 16.71
      209324_s_atRGS16Regulator of G-protein signalling 166.355
      204270_atSKIv-SKI sarcoma viral oncogene homolog (avian)6.186
      204995_atCDK5R1Cyclin-dependent kinase 5, regulatory subunit1 (p35)6.128
      204790_atSMAD7SMAD family member 75.911
      225142_atJHDM1DJumonji C domain containing histone demethylase 15.656
      205977_s_atEPHA1EPH receptor A15.258
      212680_x_atPPP1R14BProtein phosphatase 1, regulatory (inhibitor) subunit 14B5.179
      228067_atC2orf55Chromosome 2 open reading frame 555.112
      205016_atTGFATransforming growth factor, α4.895
      235851_s_atGNASGNAS complex locus4.89
      213629_x_atMT1FMetallothionein 1F (functional)4.877
      220009_atLONRF3LON peptidase N-terminal domain and ring finger 34.7
      225481_atFRMD6FERM domain containing 64.66
      213060_s_atCHI3L2Chitinase 3-like 24.64
      216268_s_atJAG1Jagged 14.51
      1565525_a_atTCP11L2t-Complex 11 (mouse)-like 24.464
      213994_s_atSPON1Spondin 1, extracellular matrix protein4.34
      201389_atITGA5Integrin, α54.316
      236241_atMED31Mediator of RNA polymerase II transcription, subunit 314.225
      203066_atCHST15Carbohydrate sulfotransferase 154.199
      223916_s_atBCORBCL6 co-repressor4.141
      231798_atNOGNoggin4.055
      205899_atCCNA1Cyclin A14.01
      212582_atOSBPL8Oxysterol binding protein-like 84
      TABLE 2The top 40 genes ranked by fold change identified as being down-regulated in T-cells following LFA-1/ICAM-1 interaction
      Probe IDSymbolGene nameFold change
      205898_atCX3CR1Chemokine (C-X3-C motif) receptor 1−17.92
      1553681_a_atPRF1Perforin1 (pore-forming protein)−8.06
      207651_atGPR171G protein-coupled receptor 171−5.92
      209840_s_atLRRN3Leucine-rich repeat neuronal 3−5.81
      1565566_a_atSTX7Syntaxin 7−5.41
      207324_s_atDSC1Desmocollin 1−5.03
      207583_atABCD2ATP-binding cassette, subfamily D (ALD), member 2−4.93
      206364_atKIF14Kinesin family member 14−4.85
      1569107_s_atZNF642Zinc finger protein 642−4.67
      236226_atBTLAB and T lymphocyte-associated−4.55
      224414_s_atCARD6Caspase recruitment domain family, member 6−4.48
      202870_s_atCDC20CDC20 cell division cycle 20−4.46
      228057_atDDIT4LDNA damage-inducible transcript 4-like−4.41
      215894_atPTGDRProstaglandin D2 receptor−4.35
      209773_s_atRRM2Ribonucleotide reductase M2 polypeptide−4.22
      207761_s_atMETTL7AMethyltransferase-like 7A−4.18
      244654_atMYO1GMyosin IG−4.18
      238581_atGBP5Guanylate-binding protein 5−4.10
      205467_atCASP10Caspase-10, apoptosis-related cysteine peptidase−4.08
      202589_atTYMSThymidylate synthetase−4.00
      218663_atNCAP-GNon-SMC condensin I complex, subunit G−3.95
      209714_s_atCDKN3Cyclin-dependent kinase inhibitor 3−3.94
      232375_atSTAT1Signal transducer and activator of transcription 1−3.70
      201291_s_atTOP2ATopoisomerase (DNA) II α 170 kDa−3.69
      1563209_a_atC20orf133Chromosome 20 open reading frame 133−3.57
      226661_atCDCA2Cell division cycle associated 2−3.55
      208059_atCCR8Chemokine (C-C motif) receptor 8−3.42
      221969_atPAX5Paired box gene5 (B-cell lineage-specific activator)−3.41
      224160_s_atACAD9Acyl-coenzyme A dehydrogenase family, member 9−3.33
      202458_atPRSS23Protease, serine, 23−3.29
      228055_atNAPSBNapsin B aspartic peptidase−3.27
      219787_s_atECT2Epithelial cell transforming sequence 2 oncogene−3.25
      202269_x_atGBP1Guanylate binding protein 1−3.11
      215358_x_atZNF37BZinc finger protein 37b (KOX 21)−3.11
      205297_s_atCD79BCD79B antigen (immunoglobulin-associated β)−2.81
      220118_atZBTB32Zinc finger and BTB domain containing 32−2.77
      235372_atFCRLAFc receptor-like A−2.75
      226453_atAYP1AYP1 protein−2.7
      226453_atRNASEH2CRibonuclease H2, subunit C−2.67
      202531_atIRF1Interferon regulatory factor 1−2.62
      In silico analysis of the identified genes was performed to interrogate associated pathways and biological networks. We applied the Pathway-Express tool of Onto-Express (using the KEGG database) to identify cellular processes affected by the identified genes. Among the sets of genes involved in various pathways influenced by LFA-1/ICAM-1 interactions were those of the TGF-β pathway, along with the genes previously identified as being involved in axon guidance, ubiquitin-mediated proteolysis, and Notch signaling. Genes associated with TGF-β related pathways particularly induced by LFA-1/ICAM-1-mediated signal included SKIL (15.48-fold), SMURF2 (9.83-fold), SMURF1 (6.71-fold), and SMAD7 (5.91-fold) (Table 1). A biological network was generated by IPA using the Ingenuity Knowledge database that demonstrate a high level of interconnectivity among these genes in TGF-β-related pathways (Fig. 1). Moreover, molecules involved in Notch signaling, including NOTCH1, JAG1, JAG2, and HEY1, were connected directly or indirectly to the identified TGF-β pathway. Because TGF-β produced by both epithelial cells and regulatory T-cells plays crucial roles in modulating an effector T-cell program (
      • Rubtsov Y.P.
      • Rudensky A.Y.
      TGFβ signaling in control of T-cell-mediated self-reactivity.
      ,
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ,
      • Li M.O.
      • Sanjabi S.
      • Flavell R.A.
      Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
      ), we elected to focus on the TGF-β-related pathway for further verification and functional analysis.
      Figure thumbnail gr1
      FIGURE 1Network pathways analysis of molecules identified in T-cells that are involved in LFA-1/ICAM-1 signaling. Each network is displayed as nodes that represent genes, and edges represent the interactions between genes. The path designer mode was used to generate final network images. Molecules shaded in green indicate a 2-fold or greater decrease in abundance, molecules shaded in red correspond to a 2-fold or greater increase in T-cells following LFA-1/ICAM-1 interactions, and the color intensity corresponds to the degree of abundance. Molecules in white are those identified through the Ingenuity Pathway Knowledgebase. The shapes denote the molecular class of the gene products. A solid line indicates a direct molecular interaction, and a dashed line indicates an indirect molecular interaction.

      Confirmation of Microarray Data by qRT-PCR

      To verify microarray data obtained from T-cells following LFA-1/ICAM-1 engagement, qRT-PCR analysis was conducted utilizing the same set of samples used for the microarray assay as well as RNA samples from independent experiments. Sixteen genes of interest (namely SMAD7, SMURF2, SKI, SKIL, NOTCH1, JAG1, JAG2, HEY1, F2RL1, CTSL, CDK5R1, RHOU, SEMA4C, EPHA1, NOG, and RGS16) with significant up-regulation were selected for qRT-PCR confirmation. Care was taken to include genes with weak (close to 2-fold) as well as strong changes (up to 50-fold) in RNA levels. qRT-PCR expression data (Fig. 2) were consistent with the results obtained by Affymetrix microarray (Table 1). Importantly, in accordance to microarray data, the mRNA levels of genes associated with the TGF-β pathway, namely SMAD7, SMURF2, SKI, and SKIL were up-regulated significantly by LFA-1/ICAM-1 signaling (Fig. 2, a–d). These results confirm a direct effect of LFA-1/ICAM-1-mediated signal on transcriptional regulation of genes involved in TGF-β signaling pathway.
      Figure thumbnail gr2
      FIGURE 2Validation of Affymatrix data for selected genes by qRT-PCR. PBL T-cells purified from healthy volunteers were incubated on ICAM-1-coated plates for 0 h (control), 1, 3, or 6 h. mRNA levels of SMAD7 (a), SMURF2 (b), SKI (c), SKIL (d), NOTCH1 (e), JAG1 (f), JAG2 (g), HEY1 (h), F2RL1 (i), CTSL (j), CDK5R1 (k), RHOU (l), SEMA4C (m), EPHA1 (n), NOG (o), and RGS16 (p) were measured by qRT-PCR. Data are fold change relative to GAPDH (mean ± S.E.) of three independent experiments. *, p < 0.05 with respect to corresponding controls.

      Up-regulation of TGF-β Signaling Proteins in LFA-1/ICAM-1-stimulated T-cells Is Mediated via STAT3 and/or JNK

      Because SMAD7, SMURF2, and SKI proteins are known to play major roles in the TGF-β signal transduction pathway (
      • Moustakas A.
      • Souchelnytskyi S.
      • Heldin C.H.
      Smad regulation in TGF-β signal transduction.
      ,
      • Nakao A.
      • Afrakhte M.
      • Morén A.
      • Nakayama T.
      • Christian J.L.
      • Heuchel R.
      • Itoh S.
      • Kawabata M.
      • Heldin N.E.
      • Heldin C.H.
      • ten Dijke P.
      Identification of Smad7, a TGFβ-inducible antagonist of TGF- β signaling.
      ,
      • Deheuninck J.
      • Luo K.
      Ski and SnoN, potent negative regulators of TGF-β signaling.
      ,
      • Schmierer B.
      • Hill C.S.
      TGFβ-SMAD signal transduction: Molecular specificity and functional flexibility.
      ,
      • Lin X.
      • Liang M.
      • Feng X.H.
      Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling.
      ,
      • Kavsak P.
      • Rasmussen R.K.
      • Causing C.G.
      • Bonni S.
      • Zhu H.
      • Thomsen G.H.
      • Wrana J.L.
      Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGFβ receptor for degradation.
      ,
      • Sun Y.
      • Liu X.
      • Eaton E.N.
      • Lane W.S.
      • Lodish H.F.
      • Weinberg R.A.
      Interaction of the Ski oncoprotein with Smad3 regulates TGF-β signaling.
      ,
      • Wu J.W.
      • Krawitz A.R.
      • Chai J.
      • Li W.
      • Zhang F.
      • Luo K.
      • Shi Y.
      Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: Insights on Ski-mediated repression of TGF-β signaling.
      ), we chose to further validate their involvement in LFA-1/ICAM-1-mediated signaling. Western immunoblot analysis showed that expression levels of SMAD7, SMURF2, and SKI were significantly increased in PBL T-cells isolated from healthy volunteers (Fig. 3a) as well as in cultured human T-cell line Hut78 (Fig. 3b) following incubation with immobilized ICAM-1 over a time period of 24 h. The expression levels of these proteins reached a plateau at 6 h and remained stable from 12 to 24 h (Fig. 3, a and b). Therefore, we used the 6-h time point for further experiments. The induction of SMAD7, SMURF2, and SKI proteins was at levels consistent in relative terms with the results obtained in the Affymetrix array and qRT-PCR analysis (Fig. 2, a–c). The up-regulation of these molecules was independent of indigenous TGF-β, as no significant change in the levels of TGF-β was detected following incubation of T-cells with immobilized ICAM-1 over 6 h (data not shown).
      Figure thumbnail gr3
      FIGURE 3Western blot analysis of LFA-1/ICAM-1-mediated change in the expression levels of SMAD7, SMURF2, and SKI proteins. PBL T-cells purified from healthy volunteers (a) or Hut78 T-cells (b) were incubated on ICAM-1-coated plates for 1, 3, 6, 12, or 24 h and lysed. c, serum-starved PBL T-cells were pretreated with 10 μm AG490, 50 μm STAT3-specific inhibitory peptide (ST3 pep), or 10 μm JNK inhibitor SP600125 (JNK-I) for 1 h and then incubated on ICAM-1-coated plated for additional 6 h. Cell lysates (20 μg each) were Western blotted and probed with anti-SMAD7, anti-SMURF2, or anti-SKI. Blots were reprobed with anti-α-tubulin or anti-β-actin as a loading control. Relative densitometric analysis of the individual protein band is presented. Data are representative of three independent experiments (mean ± S.E.). *, p < 0.05 with respect to corresponding controls.
      It has been shown previously that LFA-1 interacts with the transcriptional co-activator Jun-activation domain-binding protein 1 (JAB-1) and activates the transcription factor AP-1 through c-Jun phosphorylation (
      • Shimizu Y.
      LFA-1: More than just T cell velcro.
      ,
      • Perez O.D.
      • Mitchell D.
      • Jager G.C.
      • South S.
      • Murriel C.
      • McBride J.
      • Herzenberg L.A.
      • Kinoshita S.
      • Nolan G.P.
      Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1.
      ,
      • Bianchi E.
      • Denti S.
      • Granata A.
      • Bossi G.
      • Geginat J.
      • Villa A.
      • Rogge L.
      • Pardi R.
      Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity.
      ), which is mediated by c-Jun N-terminal kinase (JNK) (
      • Karin M.
      The regulation of AP-1 activity by mitogen-activated protein kinases.
      ,
      • Whitmarsh A.J.
      • Davis R.J.
      Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways.
      ). Furthermore, we have demonstrated recently that LFA-1 stimulation in T-cells rapidly activates a transcription factor STAT3 by tyrosine phosphorylation and subsequent nuclear translocation (
      • Verma N.K.
      • Dourlat J.
      • Davies A.M.
      • Long A.
      • Liu W.Q.
      • Garbay C.
      • Kelleher D.
      • Volkov Y.
      STAT3-stathmin interactions control microtubule dynamics in migrating T-cells.
      ). Using the Text Mining Application from SABiosciences and the UCSC Genome Browser, we identified six, three, and two binding sites for STAT3, c-Jun, and AP-1 in the promoter of SMAD7, one binding site each for c-Jun and AP-1 in SMURF2, and one binding site each for STAT3 and c-Jun in SKI, respectively. Therefore, we examined whether LFA-1/ICAM-1-mediated up-regulation of TGF-β signaling proteins was dependent on STAT3 and/or JNK activation. Although pretreatment of T-cells with a JNK-specific inhibitor partly diminished the expression of all the three TGF-β inhibitory proteins, AG490- or STAT3-specific inhibitory peptide inhibited LFA-1/ICAM-1-mediated expression of SMAD7 and SKI but not SMURF2 (Fig. 3c).

      T-cells Become Refractory to TGF-β Signaling Following LFA-1/ICAM-1 Interactions

      We next investigated whether LFA-1/ICAM-1-mediated changes in gene expression in T-cells, in particular those molecules that negatively regulate TGF-β signaling, is reflected in terms of TGF-β sensitivity. We first assayed TGF-β sensitivity of T-cells for their ability to produce IL-2. Both PBL and Hut78 T-cells secrete IL-2 following stimulation via CD3/CD28. TGF-β has been shown to suppress IL-2 production of T-cells (
      • Das L.
      • Levine A.D.
      TGF-β inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction.
      ), and as such has been proposed to be a major contributor to T-cell tolerance and immune suppression. We observed that TGF-β significantly reduced anti-CD3/CD28-stimulated IL-2 release in PBL (Fig. 4a, lane 5 versus 6) as well as in Hut78 T-cells (data not shown). However, TGF-β failed to suppress IL-2 secretion in T-cells that have been preincubated with ICAM-1 (Fig. 4a, lane 7 versus 8). Inhibiting LFA-1 signaling in T-cells by using a blocking antibody restored the ability of TGF-β to inhibit IL-2 secretion in the presence of ICAM-1 (Fig. 4a, lane 11 versus 12), which was otherwise impaired (Fig. 4a, lane 7 versus 8 and lane 9 versus 10).
      Figure thumbnail gr4
      FIGURE 4LFA-1/ICAM-1 stimulated T-cells are unresponsive to TGF-β. a, PBL T-cells purified from healthy volunteers were pre-treated with or without control IgG, or LFA-1 blocking antibody (α-LFA-1) for 30 min. These cells were stimulated with or without ICAM-1 for 6 h and then incubated with anti-CD3/CD28 (α-CD3/28) for an additional 24 h in the presence or absence of TGF-β. Conditioned medium was collected, and IL-2 secretion was measured by ELISA. b, PBL T-cells pre-treated with or without control IgG, or LFA-1 blocking antibody (α-LFA-1) were stimulated via LFA-1/ICAM-1 for 6 h or un-stimulated and then treated with or without 5 ng/ml TGF-β before lysis. Cell lysates (20 μg each) were Western blotted and probed with anti-pSMAD2 or anti-SMAD2. Relative densitometric analysis of the individual pSMAD2 protein band is presented. Data are representative of three independent experiments (mean ± S.E.). *, p < 0.05; NS, not significant.
      To further validate our findings, we investigated additional functional components regulating TGF-β signaling in T-cells influenced by LFA-1/ICAM-1 interactions. We assayed LFA-1/ICAM-1 stimulated T-cells for TGF-β sensitivity in terms of its ability to induce SMAD2 phosphorylation, a marker of TGF-β receptor activity (
      • Moustakas A.
      • Souchelnytskyi S.
      • Heldin C.H.
      Smad regulation in TGF-β signal transduction.
      ,
      • Schmierer B.
      • Hill C.S.
      TGFβ-SMAD signal transduction: Molecular specificity and functional flexibility.
      ). TGF-β treatment resulted in the increased phosphorylation of SMAD2 in both PBL (Fig. 4b, lane 2 versus 1) and in Hut78 T-cells (data not shown). However, LFA-1/ICAM-1 interactions caused inhibition of TGF-β-induced SMAD2 phosphorylation (Fig. 4b, lane 4 versus 2), indicating that T-cells became refractive to TGF-β signaling. When PBL T-cells were pretreated with LFA-1 blocking antibody, they remained sensitive to TGF-β in terms of SMAD2 phosphorylation even when incubated on immobilized ICAM-1(Fig. 4b, lane 8 versus 6).

      LFA-1/ICAM-1-mediated Up-regulation of SMAD7, SMURF2, and SKI Impairs TGF-β Signaling in T-cells

      To investigate the specific role of the identified molecules in T-cell TGF-β signaling, we utilized the well characterized Hut78 cell culture model (
      • Verma N.K.
      • Dourlat J.
      • Davies A.M.
      • Long A.
      • Liu W.Q.
      • Garbay C.
      • Kelleher D.
      • Volkov Y.
      STAT3-stathmin interactions control microtubule dynamics in migrating T-cells.
      ,
      • Volkov Y.
      • Long A.
      • Kelleher D.
      Inside the crawling T cell: Leukocyte function-asociated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isozymes β(I) and δ.
      ,
      • Verma N.K.
      • Dempsey E.
      • Conroy J.
      • Olwell P.
      • Mcelligott A.M.
      • Davies A.M.
      • Kelleher D.
      • Butini S.
      • Campiani G.
      • Williams D.C.
      • Zisterer D.M.
      • Lawler M.
      • Volkov Y.
      A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin.
      ,
      • Verma N.K.
      • Dempsey E.
      • Freeley M.
      • Botting C.H.
      • Long A.
      • Kelleher D.
      • Volkov Y.
      Analysis of dynamic tyrosine phosphoproteome in LFA-1 triggered migrating T-cells.
      ,
      • Verma N.K.
      • Davies A.M.
      • Long A.
      • Kelleher D.
      • Volkov Y.
      STAT3 knockdown by siRNA induces apoptosis in human cutaneous T-cell lymphoma line Hut78 via down-regulation of Bcl-xl.
      ), which permitted the use of plasmid constructs or siRNA to modulate specific protein expression. Transfecting T-cells with a plasmid vector containing FLAG-SMAD7 gene construct increased (up to 3-fold) the expression of SMAD7 (data not shown). SMAD7-overexpressing T-cells were unresponsive to TGF-β in terms of SMAD2 phosphorylation (Fig. 5a). Conversely, following specific knockdown (>75%) of SMAD7 in T-cells by using an siRNA approach (data not shown), there was a substantial increase in TGF-β-induced SMAD2 phosphorylation (Fig. 5b). Furthermore, SMAD7-depleted T-cells showed TGF-β-induced phosphorylation of SMAD2 even after LFA-1/ICAM-1 stimulation (Fig. 5b). In a similar manner, cells that have been electroporated with SMURF2 siRNA and stimulated through LFA-1/ICAM-1 showed a significant increase in TGF-β-induced SMAD2 phosphorylation (Fig. 5c). However, we could not detect any changes in TGF-β-induced SMAD2 phosphorylation levels in T-cells transfected with SKI siRNA as compared with their respective controls (Fig. 5d). This may be because SKI regulates TGF-β signaling downstream to SMAD2 (
      • Deheuninck J.
      • Luo K.
      Ski and SnoN, potent negative regulators of TGF-β signaling.
      ). Depletion of SMAD7, SMURF2, or SKI by siRNA in T-cells resulted in the restoration of their sensitivity to TGF-β (which was impaired by LFA-1/ICAM-1 mediated signaling) in terms of IL-2 secretion (Fig. 5e), indicating that SKI has similar functional effects albeit mediated potentially through a different mechanism.
      Figure thumbnail gr5
      FIGURE 5Specific alteration of SMAD7, SMURF2, or SKI expression modulates T-cell TGF-β responsiveness. Hut78 T-cells were electroporated with plasmid construct containing SMAD7-FLAG or vector alone (a); nonspecific siRNA (N/S) or specific siRNA against SMAD7 (b), SMURF2 (c), or SKI (d). After 48 h, cells were stimulated via LFA-1/ICAM-1 for 6 h or unstimulated and then treated with or without 5 ng/ml TGF-β for 30 min before lysis. Cell lysates (20 μg each) were Western blotted and probed with anti-pSMAD2 or anti-SMAD2. Relative densitometric analysis of the individual pSMAD2 protein band is presented. e, untransfected or siRNA (N/S, SMAD7, SMURF2, or SKI) transfected Hut78 T-cells were stimulated via LFA-1/ICAM-1 for 6 h and then incubated with anti-CD3/CD28 for additional 24 h in the presence or absence of TGF-β. Conditioned medium was collected, and IL-2 secretion was measured by ELISA. Data are representative of three independent experiments performed in triplicate (mean ± S.E.). *, p < 0.05; NS, not significant.

      LFA-1/ICAM-1-stimulated T-cells Are Refractory to TGF-β-induced iTreg or Th17 Conversion

      Given the crucial role of TGF-β signaling pathway in the T-cell differentiation program (
      • Rubtsov Y.P.
      • Rudensky A.Y.
      TGFβ signaling in control of T-cell-mediated self-reactivity.
      ,
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ,
      • Li M.O.
      • Sanjabi S.
      • Flavell R.A.
      Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
      ) and the above data indicating that LFA-1/ICAM-1-stimulated T-cells are refractory to TGF-β signaling, we examined the impact of ICAM-1/LFA-1 interactions in the development of RORγt+ Th17 or FOXP3+ regulatory T-cells. Previous studies have demonstrated that TGF-β in the presence of IL-2 can induce FOXP3 expression in human T-cell receptor activated CD4+ T-cells in vitro (
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Tran D.Q.
      • Ramsey H.
      • Shevach E.M.
      Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-β-dependent but does not confer a regulatory phenotype.
      ), a process associated with the development of a phenotype that resembles iTreg. We compared TGF-β induced FOXP3 expression between unstimulated human T-cells and cells prestimulated through LFA-1/ICAM-1 by immunostaining and high content analysis. Induction of FOXP3 expression was observed in a substantial percentage (∼50%) of anti-CD3/CD28-primed CD4+ T-cells in the presence of TGF-β (Fig. 6a, lane 2). However, FOXP3+ cell numbers in the T-cells stimulated through LFA-1/ICAM-1 were significantly lower (∼20%) than those present in unstimulated T-cells (Fig. 6a, lane 2 versus 4). To further confirm these findings, CD4+ T-cells isolated from Foxp3-eGFP reporter mice were incubated under conditions for FOXP3+ cell development in the presence or absence of ICAM-1. Flow cytometry analysis demonstrated that the frequency of FOXP3+ CD4+ T-cells was significantly (p < 0.05) impaired in the presence of ICAM-1 (Fig. 6b). TGF-β was able to induce expression of FOXP3 in >40% of CD4+ T-cells. However, in the presence of ICAM-1, a significantly smaller number (∼20%) of CD4+ T-cells could be induced to express FOXP3 (Fig. 6c, lane 2 versus 4).
      Figure thumbnail gr6
      FIGURE 6LFA-1/ICAM-1-mediated signaling in T-cells suppresses TGF-β-induced FOXP3+ iTreg or RORγt+ Th17 differentiation. a, unstimulated or LFA-1/ICAM-1 stimulated human PBL T-cells were cultured with or without 5 ng/ml TGF-β under iTreg differentiation conditions for 5 days. Cells were stained for CD4 and FOXP3 followed by high content imaging and analysis. Percentage of FOXP3+ cells among CD4+ T-cells are presented. b, representative flow cytometry plots of Foxp3-eGFP expressing CD4+ T-cells from FOXP3-eGFP reporter mice. CD4+ cells were isolated and incubated with or without ICAM-1 under iTreg conditions for 72 h in the presence or absence of 5 ng/ml TGF-β. c, mean percentage of FOXP3-eGFP+ mouse CD4+ cells treated as indicated (data are mean ± S.E. from four to six individual mice). d, unstimulated or LFA-1/ICAM-1-stimulated human PBL T-cells were cultured with or without 5 ng/ml TGF-β under Th17 conditions for 4 days. Cells were stained for CD4 and RORγt, followed by high content imaging and analysis. Percentage of RORγt+ cells among CD4+ T-cells is presented. e, expression of IL-17, detected by ELISA, in supernatants from cells cultured as described in d. Data are representative of at least three independent experiments (mean ± S.E.). *, p < 0.05.
      Recent studies have demonstrated that TGF-β in conjugation with the proinflammatory cytokine IL-6 initiates IL-17 producing RORγt+ Th17 cell development (
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ). When stimulated via CD3/CD28 in the presence of IL-6, anti-IFN-γ, and anti-IL-4 for 4 days, TGF-β induced differentiation of ∼40% CD4+ T-cells into RORγt+ cells as quantified by high content analysis (Fig. 6d, lane 2). However, pre-exposure to the LFA-1/ICAM-1 signal in T-cells substantially inhibited the induction of RORγt+ cells (Fig. 6d, lane 2 versus 4). LFA-1/ICAM-1 prestimulated T-cells that have been treated with TGF-β and IL-6 simultaneously in vitro secreted significantly less (∼50%) IL-17 as compared with unstimulated cells (Fig. 6e, lane 2 versus 4). These data suggest that T-cells stimulated through LFA-1/ICAM-1 are partially but significantly refractory to TGF-β-induced differentiation to RORγt+ Th-17 or FOXP3+ iTreg cells.

      DISCUSSION

      In this study, we addressed the role of LFA-1 signaling in the downstream regulation of gene expression in T-cells rendering them refractory to TGF-β function. We employed several strategies to investigate the functional impact of changes in gene expression induced by T-cell LFA-1/ICAM-1 interactions. Our data clearly demonstrate that LFA-1/ICAM-1 cross-linking in T-cells up-regulates a set of TGF-β inhibitory genes and proteins via activation of STAT3 and/or JNK, which results in refractoriness of T-cells to TGF-β signaling in terms of effector functions. The finding that the LFA-1/ICAM-1-mediated signaling had transcriptome level impact on the genes involved in T-cell immunoreactivity deserves special attention, in particular with regard to those genes participating in the regulation of TGF-β-mediated functions. We observed that the LFA-1/ICAM-1 interaction could trigger up-regulation of expression of SMAD7 and SMURF2 in addition to two other molecules, SKI and SKIL, all known regulators of TGF-β signaling.
      TGF-β is capable of inhibiting proinflammatory cytokines, including IL-2, in activated T-lymphocytes and plays a critical role in the local control of inflammation (
      • Das L.
      • Levine A.D.
      TGF-β inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction.
      ), at least in part through the modulation and control of regulatory T-cell responses. In this study, we have demonstrated that LFA-1/ICAM-1-stimulated T-cells exhibit defective TGF-β signaling, as measured by phospho-SMAD2 immunoreactivity and are resistant to TGF-β-mediated suppression of IL-2 secretion. Specific modulation of SMAD7 expression in T-cells by overexpression or siRNA-mediated knockdown enabled them to resist or respond to TGF-β. In previous studies, the physiological consequences of defective TGF-β signaling due to SMAD7 deregulation has been seen in a number of pathological conditions, including chronic inflammatory diseases (
      • Li M.O.
      • Sanjabi S.
      • Flavell R.A.
      Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
      ,
      • Schmierer B.
      • Hill C.S.
      TGFβ-SMAD signal transduction: Molecular specificity and functional flexibility.
      ,
      • Monteleone G.
      • Kumberova A.
      • Croft N.M.
      • McKenzie C.
      • Steer H.W.
      • MacDonald T.T.
      Blocking Smad7 restores TGF-β1 signaling in chronic inflammatory bowel disease.
      ,
      • Monteleone G.
      • Boirivant M.
      • Pallone F.
      • MacDonald T.T.
      TGF-β1 and Smad7 in the regulation of IBD.
      ,
      • Monteleone G.
      • Pallone F.
      • MacDonald T.T.
      Smad7 in TGF-β-mediated negative regulation of gut inflammation.
      ,
      • Nakao A.
      • Okumura K.
      • Ogawa H.
      Smad7: A new key player in TGF-β-associated disease.
      ,
      • Monteleone G.
      • Del Vecchio Blanco G.
      • Monteleone I.
      • Fina D.
      • Caruso R.
      • Gioia V.
      • Ballerini S.
      • Federici G.
      • Bernardini S.
      • Pallone S.
      • MacDonald T.T.
      Post-transcriptional regulation of Smad7 in the gut of patients with inflammatory bowel disease.
      ,
      • Smart N.G.
      • Apelqvist A.A.
      • Gu X.
      • Harmon E.B.
      • Topper J.N.
      • MacDonald R.J.
      • Kim S.K.
      Conditional expression of Smad7 in pancreatic β cells disrupts TGF-β signaling and induces reversible diabetes mellitus.
      ,
      • Boirivant M.
      • Pallone F.
      • Di Giacinto C.
      • Fina D.
      • Monteleone I.
      • Marinaro M.
      • Caruso R.
      • Colantoni A.
      • Palmieri G.
      • Sanchez M.
      • Strober W.
      • MacDonald T.T.
      • Monteleone G.
      Inhibition of Smad7 with a specific antisense oligonucleotide facilitates TGF-β1-mediated suppression of colitis.
      ). For example, in vivo administration of Smad7 antisense oligonucleotides to colitic mice restored TGF-β signaling, ameliorated inflammation, and decreased the synthesis of inflammatory molecules and the extent of gut damage (
      • Monteleone G.
      • Boirivant M.
      • Pallone F.
      • MacDonald T.T.
      TGF-β1 and Smad7 in the regulation of IBD.
      ,
      • Boirivant M.
      • Pallone F.
      • Di Giacinto C.
      • Fina D.
      • Monteleone I.
      • Marinaro M.
      • Caruso R.
      • Colantoni A.
      • Palmieri G.
      • Sanchez M.
      • Strober W.
      • MacDonald T.T.
      • Monteleone G.
      Inhibition of Smad7 with a specific antisense oligonucleotide facilitates TGF-β1-mediated suppression of colitis.
      ). Thus, blocking SMAD7 could be a promising way to dampen chronic inflammation, particularly during active phases when expression levels of SMAD7 are high.
      We also identified ubiquitination regulatory factors SMURF1 and SMURF2 to be up-regulated by LFA-1/ICAM-1 interaction in T-cells. SMURF1 and SMURF2 are members of the Hect family of E3 ubiquitin ligases that participate in the degradation of TGF-β receptors and other targets (
      • Lin X.
      • Liang M.
      • Feng X.H.
      Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling.
      ) and are involved in down-regulation of the TGF-β signaling pathway (
      • Kavsak P.
      • Rasmussen R.K.
      • Causing C.G.
      • Bonni S.
      • Zhu H.
      • Thomsen G.H.
      • Wrana J.L.
      Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGFβ receptor for degradation.
      ). SMURF2, synergistically with SMAD7 through direct interaction, interferes with the activation of receptor associated SMAD2 and SMAD3 and plays a role in their targeted degradation by ubiquitination (
      • Lin X.
      • Liang M.
      • Feng X.H.
      Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling.
      ,
      • Kavsak P.
      • Rasmussen R.K.
      • Causing C.G.
      • Bonni S.
      • Zhu H.
      • Thomsen G.H.
      • Wrana J.L.
      Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGFβ receptor for degradation.
      ). A more recent study established a crucial role of ubiquitination process involving an E2 ubiquitin-conjugating enzyme Ubc13 in maintaining the in vivo immunosuppressive function of Treg cells and in preventing the conversion of Treg into Th1- or Th17-like effector T-cells (
      • Chang J.H.
      • Xiao Y.
      • Hu H.
      • Jin J.
      • Yu J.
      • Zhou X.
      • Wu X.
      • Johnson H.M.
      • Akira S.
      • Pasparakis M.
      • Cheng X.
      • Sun S.C.
      Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells.
      ). The SKI family of nuclear oncoproteins bind directly to the SMAD3/4 complex in the nucleus and negatively regulate TGF-β signaling (
      • Sun Y.
      • Liu X.
      • Eaton E.N.
      • Lane W.S.
      • Lodish H.F.
      • Weinberg R.A.
      Interaction of the Ski oncoprotein with Smad3 regulates TGF-β signaling.
      ,
      • Wu J.W.
      • Krawitz A.R.
      • Chai J.
      • Li W.
      • Zhang F.
      • Luo K.
      • Shi Y.
      Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: Insights on Ski-mediated repression of TGF-β signaling.
      ). Although the exact role of these molecules in LFA-1/ICAM-1-stimulated T-cells under in vivo conditions remain unclear, we believe that up-regulation of potent TGF-β signaling inhibitors in T-lymphocytes would generate TGF-β unresponsiveness.
      TGF-β is a critical differentiation factor for the generation of both Th17 as well as iTreg cell subsets (
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ,
      • Li M.O.
      • Sanjabi S.
      • Flavell R.A.
      Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
      ). Although implicated in antagonistic functions, both iTregs and Th17 effector cells play crucial roles in immunoregulation, host defense, and autoimmune pathogenesis. Although Tregs play a fundamental role in protection from autoimmunity, their differentiation is tightly linked to the development of Th17 cells, a highly pathogenic effector T-cell subset involved in inducing inflammation and autoimmune tissue injury (
      • Yamagiwa S.
      • Gray J.D.
      • Hashimoto S.
      • Horwitz D.A.
      A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.
      ,
      • Mangan P.R.
      • Harrington L.E.
      • O'Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • Hatton R.D.
      • Wahl S.M.
      • Schoeb T.R.
      • Weaver C.T.
      Transforming growth factor-β induces development of the TH17 lineage.
      ,
      • Weaver C.T.
      • Hatton R.D.
      Interplay between the TH17 and TReg cell lineages: A (co-)evolutionary perspective.
      ). Recently, a subset of FOXP3+ peripheral Tregs that exhibit dual Treg and Th17 function depending on environmental factors under inflammatory conditions has also been identified (
      • Voo K.S.
      • Wang Y.H.
      • Santori F.R.
      • Boggiano C.
      • Wang Y.H.
      • Arima K.
      • Bover L.
      • Hanabuchi S.
      • Khalili J.
      • Marinova E.
      • Zheng B.
      • Littman D.R.
      • Liu Y.J.
      Identification of IL-17-producing FOXP3+ regulatory T cells in humans.
      ). Utilizing multiple approaches, we showed that human PBL T-cells as well as CD4+ T-cells isolated from transgenic reporter mice stimulated through LFA-1/ICAM-1 were resistant to TGF-β-mediated FOXP3+ iTreg induction. In a similar manner, LFA-1/ICAM-1-stimulated T-cells were refractory to TGF-β-dependent development of RORγt+ IL-17 producing Th17 cells. Previous in vivo studies have shown that mice defective in TGF-β signaling lack Th17 cells and do not develop experimental autoimmune encephalomyelitis (
      • Veldhoen M.
      • Hocking R.J.
      • Flavell R.A.
      • Stockinger B.
      Signals mediated by transforming growth factor-β initiate autoimmune encephalomyelitis, but chronic inflammation is needed to sustain disease.
      ). The observed suppressive role of LFA-1/ICAM-1-mediated signaling on TGF-β-induced iTreg as well as Th17 cell differentiation leaves open the possibility that LFA-1/ICAM-1 interaction selectively triggers gene expression, which controls the development of both lineages and thereby modulates the associated downstream functions of T-cells.
      The present study thus elucidates key biochemical and molecular mechanisms by which LFA-1/ICAM-1 interactions render T-cells refractory to TGF-β signaling. Based on these data, we now propose a model for the molecular crosstalk between LFA-1 and TGF-β signaling pathways (Fig. 7). LFA-1/ICAM-1 interactions play a key role(s) in up-regulating several molecules, including SMAD7, SMURF2, and SKI protein expression via activation of transcriptional regulators such as STAT3 and/or JNK. These molecules, in turn, render T-cells refractory to TGF-β. LFA-1/ICAM-1-mediated signaling has a profound regulatory impact on the TGF-β responsiveness of T-cells with regard to IL-2 secretion and influences the relative development of both RORγt+ Th17 and FOXP3+ iTreg lineages, possibly depending on the co-stimulated cytokine milieu in distinct tissue. Our study also suggest that the behavior of iTreg cells may be influenced in an inflamed environment characterized by T-cell migration, which may be an important consideration as Treg-based immunotherapy is making its way from bench to bedside.
      Figure thumbnail gr7
      FIGURE 7A model for LFA-1/ICAM-1-mediated signal resulting in T-cells refractory to TGF-β signaling. The figure illustrates that LFA-1/ICAM-1 interaction induces up-regulation of SMAD7, SMURF2, and SKI protein expression in T-cells via STAT3 and/or JNK (left panel). The increased expression of SMAD7, SMURF2, and SKI proteins results in T-cells refractive to TGF-β signaling and interferes with TGF-β-mediated inhibition of IL-2 secretion and T-cell differentiation processes (right panel).
      Recent in vitro, in vivo and preclinical studies in patients with stable psoriasis clearly indicate LFA-1 involvement in modulating T-cell functions, including activation and proliferation (
      • Koszik F.
      • Stary G.
      • Selenko-Gebauer N.
      • Stingl G.
      Efalizumab modulates T cell function both in vivo and in vitro.
      ,
      • Reisman N.M.
      • Floyd T.L.
      • Wagener M.E.
      • Kirk A.D.
      • Larsen C.P.
      • Ford M.L.
      LFA-1 blockade induces effector and regulatory T-cell enrichment in lymph nodes and synergizes with CTLA-4Ig to inhibit effector function.
      ). For example, in a murine transplant model, an increased frequency of FOXP3+ Tregs was observed in the peripheral lymph nodes following LFA-1 blockade by anti-LFA-1 (
      • Josefowicz S.Z.
      • Niec R.E.
      • Kim H.Y.
      • Treuting P.
      • Chinen T.
      • Zheng Y.
      • Umetsu D.T.
      • Rudensky A.Y.
      Extrathymically generated regulatory T cells control mucosal TH2 inflammation.
      ). A humanized anti-LFA-1 antibody efalizumab (Raptiva®), developed for the treatment of psoriasis, was found to induce a unique state of T-cell hyporesponsiveness in terms of activation and proliferation (
      • Koszik F.
      • Stary G.
      • Selenko-Gebauer N.
      • Stingl G.
      Efalizumab modulates T cell function both in vivo and in vitro.
      ,
      • Guttman-Yassky E.
      • Vugmeyster Y.
      • Lowes M.A.
      • Chamian F.
      • Kikuchi T.
      • Kagen M.
      • Gilleaudeau P.
      • Lee E.
      • Hunte B.
      • Howell K.
      • Dummer W.
      • Bodary S.C.
      • Krueger J.G.
      Blockade of CD11a by efalizumab in psoriasis patients induces a unique state of T-cell hyporesponsiveness.
      ). LFA-1 deficiency was also suggested to dampen encephalomyelitis upon active induction of an autoimmune response (
      • Wang Y.
      • Kai H.
      • Chang F.
      • Shibata K.
      • Tahara-Hanaoka S.
      • Honda S.
      • Shibuya A.
      • Shibuya K.
      A critical role of LFA-1 in the development of Th17 cells and induction of experimental autoimmune encephalomyelitis.
      ). Unfortunately, despite encouraging results using LFA-1 blockade, further clinical trials have been hampered by several cases of the development of progressive multi-focal leukoencephalopathy in patients receiving long term courses of efalizumab (
      • Carson K.R.
      • Focosi D.
      • Major E.O.
      • Petrini M.
      • Richey E.A.
      • West D.P.
      • Bennett C.L.
      Monoclonal antibody-associated progressive multifocal leucoencephalopathy in patients treated with rituximab, natalizumab, and efalizumab: A review from the Research on Adverse Drug Events and Reports (RADAR) Project.
      ). In the current study, interruption of LFA-1/ICAM-1 interaction by antibody blockade of LFA-1 prevented the development of TGF-β unresponsive phenotypes in T-cells. We therefore propose that signals emanating from LFA-1/ICAM-1 interaction fine-tune classical immune suppression by TGF-β, possibly facilitating the delivery of effector lymphocytes primed to respond at the site of inflammation. It can be speculated that drugs that target LFA-1 (such as efalizumab) could also generate some of their functional effects through increasing T-cell responsiveness to TGF-β and thus modulate the Treg phenotype in addition to effects on the immune synapse and on lymphocyte migration. Dissection of differential signaling for migration and for generation of TGF-β hyporesponsiveness might permit the development of more selective inhibitors of LFA-1 with less pronounced effects on immune responsiveness to viruses such as the JC-1 virus implicated in progressive multi-focal leukoencephalopathy. These findings indicate the need for judicious application of LFA-1 targeting and may have implications for designing of immunotherapies in the clinical settings.

      Acknowledgments

      We thank Prof. Carl-Henrik Heldinm (Ludwig Institute for Cancer Research Ltd., Uppsala University, Uppsala, Sweden) for providing the SMAD7 plasmid construct as a gift. We thank Dara Dunican and Michael Freeley for sharing experiences in experimentation and Anne Murphy for technical assistance.

      Supplementary Material

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