Human OX40 tunes the function of regulatory T cells in tumor and nontumor areas of hepatitis C virus-infected liver tissue

HepatologyVolume 60, Issue 5 p. 1494-1507 Viral HepatitisFree Access Human OX40 tunes the function of regulatory T cells in tumor and nontumor areas of hepatitis C virus–infected liver tissue Silvia Piconese, Silvia Piconese Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy Equally contributing authors.Search for more papers by this authorEleonora Timperi, Eleonora Timperi Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy Equally contributing authors.Search for more papers by this authorIlenia Pacella, Ilenia Pacella Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorValeria Schinzari, Valeria Schinzari Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorClaudio Tripodo, Claudio Tripodo Dipartimento di Scienze per la Promozione della Salute, Università degli Studi di Palermo, Palermo, ItalySearch for more papers by this authorMassimo Rossi, Massimo Rossi Dipartimento di Chirurgia Generale e Trapianti d'Organo, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorNicola Guglielmo, Nicola Guglielmo Dipartimento di Chirurgia Generale e Trapianti d'Organo, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorGianluca Mennini, Gianluca Mennini Dipartimento di Chirurgia Generale e Trapianti d'Organo, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorGian Luca Grazi, Gian Luca Grazi Chirurgia Epato-bilio-pancreatica, Istituto Nazionale dei Tumori “Regina Elena”, Rome, ItalySearch for more papers by this authorSimona Di Filippo, Simona Di Filippo Chirurgia Epato-bilio-pancreatica, Istituto Nazionale dei Tumori “Regina Elena”, Rome, ItalySearch for more papers by this authorStefania Brozzetti, Stefania Brozzetti Dipartimento di Chirurgia “Pietro Valdoni,”, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorKatia Fazzi, Katia Fazzi Dipartimento di Chirurgia “Pietro Valdoni,”, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorGuido Antonelli, Guido Antonelli Laboratorio di Virologia, Dipartimento di Medicina Molecolare, “Sapienza” Università di Roma, Rome, ItalySearch for more papers by this authorMaria Antonietta Lozzi, Maria Antonietta Lozzi Laboratorio di Virologia, Dipartimento di Medicina Molecolare, “Sapienza” Università di Roma, Rome, ItalySearch for more papers by this authorMassimo Sanchez, Massimo Sanchez Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorVincenzo Barnaba, Corresponding Author Vincenzo Barnaba Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy Istituto Pasteur–Fondazione Cenci Bolognetti, Rome, Italy Fondazione Andrea Cesalpino, Rome, ItalyAddress reprint requests to: Vincenzo Barnaba, M.D., “Sapienza” Università di Roma, Viale del Policlinico 155, 00161 Rome, Italy. E-mail: vincenzo.barnaba@uniroma1.it; Fax: +39 (6) 49383333.Search for more papers by this author Silvia Piconese, Silvia Piconese Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy Equally contributing authors.Search for more papers by this authorEleonora Timperi, Eleonora Timperi Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy Equally contributing authors.Search for more papers by this authorIlenia Pacella, Ilenia Pacella Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorValeria Schinzari, Valeria Schinzari Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorClaudio Tripodo, Claudio Tripodo Dipartimento di Scienze per la Promozione della Salute, Università degli Studi di Palermo, Palermo, ItalySearch for more papers by this authorMassimo Rossi, Massimo Rossi Dipartimento di Chirurgia Generale e Trapianti d'Organo, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorNicola Guglielmo, Nicola Guglielmo Dipartimento di Chirurgia Generale e Trapianti d'Organo, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorGianluca Mennini, Gianluca Mennini Dipartimento di Chirurgia Generale e Trapianti d'Organo, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorGian Luca Grazi, Gian Luca Grazi Chirurgia Epato-bilio-pancreatica, Istituto Nazionale dei Tumori “Regina Elena”, Rome, ItalySearch for more papers by this authorSimona Di Filippo, Simona Di Filippo Chirurgia Epato-bilio-pancreatica, Istituto Nazionale dei Tumori “Regina Elena”, Rome, ItalySearch for more papers by this authorStefania Brozzetti, Stefania Brozzetti Dipartimento di Chirurgia “Pietro Valdoni,”, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorKatia Fazzi, Katia Fazzi Dipartimento di Chirurgia “Pietro Valdoni,”, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, ItalySearch for more papers by this authorGuido Antonelli, Guido Antonelli Laboratorio di Virologia, Dipartimento di Medicina Molecolare, “Sapienza” Università di Roma, Rome, ItalySearch for more papers by this authorMaria Antonietta Lozzi, Maria Antonietta Lozzi Laboratorio di Virologia, Dipartimento di Medicina Molecolare, “Sapienza” Università di Roma, Rome, ItalySearch for more papers by this authorMassimo Sanchez, Massimo Sanchez Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorVincenzo Barnaba, Corresponding Author Vincenzo Barnaba Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy Istituto Pasteur–Fondazione Cenci Bolognetti, Rome, Italy Fondazione Andrea Cesalpino, Rome, ItalyAddress reprint requests to: Vincenzo Barnaba, M.D., “Sapienza” Università di Roma, Viale del Policlinico 155, 00161 Rome, Italy. E-mail: vincenzo.barnaba@uniroma1.it; Fax: +39 (6) 49383333.Search for more papers by this author First published: 23 April 2014 https://doi.org/10.1002/hep.27188Citations: 60 Potential conflict of interest: Prof. Antonelli received grants from Siemens and Vin. This work was supported by the following grants obtained by V.B.: Associazione Italiana per la Ricerca sul Cancro (AIRC; progetto “Investigator Grant” [IG]-2010/13 no. 10756); European Union grants (IMECS no. 201169, FP7-Health-2007-A, and SPHYNX no. 261365, FP7-Health-2010); Ministero della Sanità (Ricerca finalizzata [RFPS-2006-3-337923 and RFPS-2007-1-636647] and Istituto Superiore di Sanità [Progetto AIDS-2008]); Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR; Programmi di ricerca di interesse nazionale [PRIN]-2008/10 no. 7245/1; [PRIN]-2011/13 no. 2010LC747T-004; Ateneo Sapienza [2009-C26A09PELN, 2010-C26A1029ZS, 2011-C26A11BYWP, and 2012-C26A12JL55]; and Fondo per gli investimenti di ricerca di base [FIRB]-2011/13 no. RBAP10TPXK); Fondazione Cariplo (progetti no. 13535 and 3603 2010/12); FISM (Fondazione Italiana Sclerosi Multipla onlus) grant no. 2011/R/4; Fondazione Italiana per la Ricerca sull'Artrite (FIRA 2010); and Istituto Italiano di Tecnologia (IIT; A2 project 2013). This work was also supported by grants obtained by S.P. from Associazione Italiana Ricerca sul Cancro (MFAG 8726) and from Ministero dell'Istruzione, dell'Università e della Ricerca (FIRB-Futuro in ricerca RBFR12I3UB_002). See Editorial on Page 1461 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Regulatory T cells (Tregs) can be considered as a mixed population of distinct subsets, endowed with a diverse extent and quality of adaptation to microenvironmental signals. Here, we uncovered an opposite distribution of Treg expansion, phenotype, and plasticity in different microenvironments in the same organ (liver) derived from patients with chronic hepatitis C: On the one side, cirrhotic and tumor fragments were moderately and highly infiltrated by Tregs, respectively, expressing OX40 and a T-bethighIFN-γ– “T-helper (Th)1-suppressing” phenotype; on the other side, noncirrhotic liver specimens contained low frequencies of Tregs that expressed low levels of OX40 and highly produced interferon-gamma (IFN-γ; T-bet+IFN-γ+), thus becoming “Th1-like” cells. OX40-expressing and Th1-suppressing Tregs were enriched in the Helios-positive subset, carrying highly demethylated Treg cell-specific demethylated region that configures committed Tregs stably expressing forkhead box protein 3. OX40 ligand, mostly expressed by M2-like monocytes and macrophages, boosted OX40+ Treg proliferation and antagonized the differentiation of Th1-like Tregs. However, this signal is counteracted in noncirrhotic liver tissue (showing various levels of inflammation) by high availability of interleukin-12 and IFN-γ, ultimately leading to complete, full Th1-like Treg differentiation. Conclusion: Our data demonstrate that Tregs can finely adapt, or even subvert, their classical inhibitory machinery in distinct microenvironments within the same organ. (Hepatology 2014;60:1494–1507) Abbreviations Ab antibody act activated c cirrhotic AFLD alcoholic fatty liver disease CFSE carboxyfluorescein succinimidyl ester CHC chronic hepatitis C DCs dendritic cells FCM flow cytometry FOXP3 forkhead box protein 3 HCC hepatocellular carcinoma HCV hepatitis C virus HD healthy donor IFN-γ interferon-gamma IHC immunohistochemical IL interleukin LPS lipopolysaccharide MFI mean fluorescence intensity MNCs mononuclear cells mono/MF monocytes/macrophages nc noncirrhotic NT-LIV nontumoral liver PB peripheral blood PD-1 programmed death-1 pts patients r recombinant Tconv conventional T cell Th T helper TNF-α tumor necrosis factor alpha Treg regulatory T cell TSDR Treg cell-specific demethylated region TUM tumor Regulatory T cells (Tregs) represent crucial gatekeepers for the maintenance of immune homeostasis and the protection from uncontrolled immune responses.1 The Treg lineage is determined by the transcription factor, forkhead box protein 3 (FOXP3), and by epigenetic, FOXP3-independent events.2 In particular, Treg stability is associated with a demethylated form of the Treg cell-specific demethylated region (TSDR) in the Foxp3 locus.3 Among the several molecules suggested to regulate Treg expansion and/or suppressive function, programmed death-1 (PD-1) and OX40 have recently received particular attention. PD-1, considered as an inhibitory receptor delivering negative signals to conventional T cells (Tconvs), sustains development, maintenance, and suppressive function of peripherally induced Tregs upon engagement with its own ligand (PD-L1).4 On the other side, PD-1 tempers expansion and function of already established Tregs.5, 6 Contrary to PD-1, OX40 is considered as a costimulatory molecule conveying prosurvival signals to Tconvs.7 OX40 is constitutively expressed by murine Tregs and inhibits Treg suppressive function,8-10 a property that has been exploited to induce antitumor response in mouse models8, 11, 12 and even in cancer patients.13 However, in some contexts, OX40 signal can foster Treg fitness and proliferation.14-17 OX40 is expressed by human Tregs or Tconvs not constitutively, but only upon appropriate activating signals7: Whether this inducible form of OX40 delivers prosurvival or inhibitory signals in human Tregs is not completely clear. Under precise microenvironment-driven signals and in pathological processes dominated by a certain T-helper (Th) subset (Th1, Th2, Th17, and TFH), Tregs (or Treg subsets) can express the corresponding T-helper-associated transcription factor(s), thus acquiring specialized suppressive functions selectively directed against that T-helper subset.18 Unstable Tregs, functionally and/or molecularly “deprogrammed” into cytokine-producing (i.e., “Th1-like” or “Th17-like”) Tregs, likely contribute to, rather than suppress, inflammatory responses.19 Whether Treg heterogeneity and plasticity are modulated in diverse pathologic liver microenvironments, and whether expression or engagement of costimulatory and inhibitory receptors is involved in Treg adaptation to microenvironmental cues, is mostly unknown. Here, we show that, within a certain organ (i.e., the human liver from CHC patients [pts]), inflammatory, cirrhosis, or tumor compartments were characterized by the expansion of distinct Treg subsets, expressing different levels of OX40, Helios (a transcription factor previously associated with Treg molecular and functional stability20) and TSDR methylation, and displaying divergent specialization or reprogramming in response to cytokines and surface signals provided by the different microenvironments. Patients and Methods Patients, Samples, and Processing 29 CHC patients with or without hepatocellular carcinoma (HCC) were studied (Supporting Table 1): 23 with underlying cirrhosis (c), 6 without cirrhosis (noncirrhotic, nc). Peripheral blood mononuclear cells (PBMCs) and liver specimens were obtained from patients undergoing surgery or liver transplantation at Istituto Nazionale dei Tumori “Regina Elena” or “Sapienza” Università di Roma - Policlinico Umberto I. Human studies have been performed in accordance to the ethical guidelines of the 1975 Declaration of Helsinki and were approved by each Institutional Ethical Committee. Informed consent was obtained from all patients. Methods for HCV-RNA viral load assessment, PBMC isolation and liver fragment processing are detailed in the Supporting Information. Flow Cytometry A complete list of antibodies can be found in the Supporting Information. Cells were pretreated with human Fc-Receptor Binding Inhibitor (eBioscience) and incubated 20 minutes at 4°C with Abs for surface antigens, except anti-OX40L and IL-12R-b2 PE (30 minutes at RT). Intracellular staining for FOXP3 was performed using the anti-FOXP3 mAb and FOXP3/Transcription Factor Staining Buffer Set according to manufacturer's instructions (eBioscience). Before IFN-γ and T-bet staining, cells were stimulated 4 hours with Cell Stimulation Cocktail (plus protein transport inhibitors, eBioscience). For mono/MF cytokine production, mononuclear cells were stimulated 18 hours with LPS (400 ng/ml, Difco) alone or plus IFN-γ (50 ng/ml, eBioscience), in the presence of Protein Transport Inhibitor Cocktail (eBioscience), and intracellular staining for TNF-α and IL-10 was performed in gated CD14+ cells using Cytofix/Cytoperm and Perm/Wash buffers according to manufacturer's instructions (BD Bioscience). Data were acquired on LSR Fortessa (Becton Dickinson) and analyzed with FlowJo software (Tree Star Inc, version 8.8.7). In Vitro Assays of Treg Proliferation, Th1-Like Polarization or Suppression Tregs were enriched using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec). CFSE labeling was performed by incubation for 15 minutes at 37°C with 10 μM CFSE. In some experiments, Tregs were preactivated for 18 hours in U-bottomed 96-well plate with equal numbers of T cell-stimulating beads (Treg Suppression Inspector, Miltenyi Biotec) plus IL-2 (100 IU/ml, Roche) and TNF-α (50 ng/ml, RnDSystems). OX40 was stimulated with rOX40L (20 ng/ml, containing a polyHistidine tag, RnDSystems) cross-linked with a secondary tag-specific mAb (10 mg/ml, AD1.1.10, mouse IgG1, RnDSystems). Details of T-cell culture and cytokine stimulation, and of Th1-like Treg polarization ex vivo, can be found in the Supporting Information. TSDR Demethylation Analysis Helioshigh and Helioslow subsets in act-Treg or non-Treg gates were sorted using a FACSAria (Becton Dickinson), and real-time PCR using methylation- and demethylation-specific primers was performed on bisulfite-treated genomic DNA, as detailed in the Supporting Information. IHC Immunohistochemical analyses were performed on formalin-fixed paraffin embedded HCC tissue samples (n = 8) obtained from the archives of the Human Pathology Unit of the Department of Health Science, University of Palermo. Antibodies and methods are described in the Supporting Information. Statistical Analysis Statistical analysis was performed using Prism software (version 4, GraphPad). Unpaired Student t test, 2-tailed, was used to analyze in vitro data, while Mann-Whitney test, 2-tailed, or Wilcoxon matched pairs test, 2-tailed, was applied to compare groups of ex vivo samples. Correlations have been calculated using the nonparametric Spearman's correlation test, 2-tailed. Every in vitro assay was performed in triplicates or quadruplicates when possible. In all graphs, bars show means ± SEM. In all tests, P < 0.05 was considered statistically significant. Results Tregs Are Differentially Distributed in Distinct Liver Microenvironments in Line With Variable OX40 Expression First, we quantified the percentage of FOXP3+CD127low Tregs,21 within the CD4+ T-cell pool, in mononuclear cells (MNCs) isolated from nontumor liver (NT-LIV) or hepatocellular carcinoma (HCC; tumor [TUM]) fragments, derived from the same CHC livers, carrying underlying cirrhosis (c) or not (nc; Supporting Table 1), as well as from peripheral blood (PB) of the same pts or healthy donors (HDs), as a control. Confirming previous studies,22, 23 Tregs were significantly expanded at the TUM site compared to the other districts (Fig. 1). Interestingly, Tregs were significantly enriched in NT-LIV and in PB of c, compared to nc, pts (Fig. 1). Such cirrhosis-related Treg expansion was peculiar to CHC, because an unaltered or even decreased Treg frequency was detected in NT-LIVc specimens obtained from pts with hepatitis B or alcoholic fatty liver disease (AFLD) pts (data not shown). Figure 1Open in figure viewerPowerPoint Tregs are significantly enriched in tumor and nontumor cirrhotic tissues. Treg frequency was estimated by FCM as the percentage of FOXP3+CD127low cells among CD4 T cells, in PB of HD, or PB, NT-LIV or TUM fragments obtained from CHC pts, c, or nc, listed in Supporting Table 1. Representative FCM data (left) and data overview (right) are shown. *P < 0.05; **P < 0.01; ***P < 0.005; by Mann-Whitney's test, two-tailed. Importantly, the frequency of OX40+ Tregs was significantly higher (compared to PB) in NT-LIVc and (at higher levels) TUM, but not in NT-LIVnc, irrespective of PD-1 expression (Fig. 2A). OX40 was also expressed in a low fraction of Tconvs (CD4+FOXP3–) from TUM and NT-LIVc, albeit at a greatly lower degree than in Tregs from the same districts (Fig. 2A). In addition, OX40+ Tregs also expressed OX40 at a higher level (in terms of mean fluorescence intensity [MFI]) than the Tconv counterpart (Supporting Fig. 1). Instead, the frequency of PD-1+OX40– cells was similar between Tconvs and Tregs in each district; in both Tconvs and Tregs, the PD-1+OX40– subset was significantly enriched in the liver, compared to PB, but irrespective of whether infiltrating NT-LIVc, NT-LIVnc, or TUM specimens (Fig. 2A). Notably, total Treg frequency was directly correlated with the percentage of OX40+ Tregs in TUM and tended to be inversely associated with the percentage of PD-1+OX40– Tregs (Fig. 2B), suggesting OX40 as a receptor associated to, or even functionally involved in, Treg expansion in cirrhosis and cancer, and PD-1 as a possible negative regulator of Treg expansion, as previously demonstrated in chronically hepatitis C virus (HCV)-infected settings.6 Figure 2Open in figure viewerPowerPoint OX40 is highly expressed by Tregs in tumor and nontumor cirrhotic tissues. (A) Frequencies of PD-1–OX40+, PD-1+OX40+, and PD-1+OX40– cells were evaluated by FCM in CD4+FOXP3– Tconvs or CD4+FOXP3+CD127low Tregs, in different samples (PB, NT-LIV, and TUM) obtained from CHC pts. Data overview (left) or representative FCM data (right) are shown. *P < 0.05; **P < 0.01; ***P < 0.005; by Mann-Whitney's test, two-tailed. (B) Spearman's correlation (r) between frequency of Tregs and frequency of OX40+ (PD-1+/–) or PD-1+OX40– Tregs in different liver specimens from CHC pts. *P < 0.05; ns, not significant. Th1-Suppressing and Th1-Like Tregs Show Phenotypic, Functional, and Spatial Dichotomy in Liver Many data demonstrate that, in contexts characterized by a Th1-oriented inflammation, Tregs become specialized Th1-suppressing cells (gaining T-bet expression) or even become Th1-like cells (acquiring the competence for interferon-gamma [IFN-γ] production), thus inhibiting or contributing to inflammation, respectively.24 Within both Tconvs and Tregs, two subpopulations could be clearly detected in the different liver districts (upon short stimulation with a phorbol myristate acetate/ionomycin-containing reagent): (1) T-bet+IFN-γ+ cells, representing cells fully differentiated into Th1 Tconvs or Th1-like Tregs, and (2) T-bethighIFN-γ– cells, representing cells undergoing an incomplete Th1 differentiation, that, in the case of Tregs, have been identified as the specialized Th1-suppressing cells24, 25 (Fig. 3A). However, the T-bet+IFN-γ+ Treg or Tconv (Th1-like or Th1, respectively) subsets were maximally expanded in NT-LIVnc and less represented in NT-LIVc and TUM (Fig. 3B, upper plots). Conversely, the T-bethighIFN-γ– subpopulation of Tregs (Th1-suppressing), but not of Tconvs, was expanded in NT-LIVc and (to a significant extent) in TUM (Fig. 3B, lower plots). Th1-like (T-bet+IFN-γ+), but not Th1-suppressing (T-bethighIFN-γ–), Treg frequencies positively correlated with the frequency of Th1 Tconvs in PB, NT-LIVc, and TUM (Fig. 3C), indicating that a Th1-oriented microenvironment may favor the polarization of Th1-like Tregs. Importantly, OX40 was differentially expressed in the two subsets, being the OX40+ Treg percentage significantly higher in the Th1-suppressing T-bethighIFN-γ– subset, compared to T-bet+IFN-γ+ Th1-like counterpart, in each sample (Fig. 3D), suggesting that OX40 may be preferentially associated with an activated and specialized suppressive function of Tregs in vivo. Figure 3Open in figure viewerPowerPoint Th1-like and -suppressing Tregs accumulate in noncirrhotic liver or in cirrhotic/tumor liver, respectively. (A) Representative intracellular staining of IFN-γ versus T-bet ex vivo in gated Tregs and Tconvs from PB or NT-LIVnc of a CHC pt. (B) Frequency of T-bet+IFN-γ+ (Th1-like) or T-bethighIFN-γ– (Th1-suppressing) in gated Tregs (left plots), and analogous subsets in gated Tconvs (right plots), from different specimens. *P < 0.05 by Mann-Whitney's test, two-tailed. (C) Spearman's correlation (r) between frequencies of T-bet+IFN-γ+ (Th1-like), or of T-bethighIFN-γ– (Th1-suppressing) Tregs, and frequency of T-bet+IFN-γ+ (Th1) Tconvs in different specimens from CHC pts. *P < 0.05; **P < 0.01; ns, not significant; na, not available. (D) Percentage of OX40+ cells in gated T-bet+IFN-γ+ (Th1-like) versus T-bethighIFN-γ– (Th1-suppressing) Tregs in each TUM sample from CHC patients. *P < 0.05 by Wilcoxon's matched pairs test, two-tailed. To ascertain the role of OX40 in human Treg-suppressive function, we set up an in vitro experimental assay by using Tregs enriched from PB of HDs (because of the paucity of liver-derived MNCs that did not allow the availability of high numbers of purified Treg required for these experiments) and preactivated in vitro for 18 hours with T-cell-stimulating beads, interleukin (IL)-2, and tumor necrosis factor alpha (TNF-α) to induce OX40 up-regulation.26 Such preactivated Tregs were more suppressive than fresh Tregs in a standard in vitro suppression assay, performed by coculturing Tregs with scaled dilutions of carboxyfluorescein succinimidyl ester (CFSE)-labeled Tconvs in the presence of irradiated peripheral blood mononuclear cells and anti-CD3 (Supporting Fig. 2A). Strikingly, highly purified OX40+ Tregs, isolated from preactivated Tregs, were significantly more suppressive than the OX40– counterpart (Supporting Fig. 2B). To test whether OX40 engagement by an agonistic molecule inhibited the suppressive function of human Tregs, as previously observed in mice,9, 10, 12 we performed experiments in which preactivated OX40-expressing Tregs, obtained as described above, were incubated for 6 hours with cross-linked rOX40L and then added to the suppression assay. In this setting, we could not detect any change in Treg-suppressive function in vitro after OX40 engagement (Supporting Fig. 2C). Altogether, these data suggest that OX40 signal neither improves nor diminishes the suppressive ability of human Tregs on a per cell basis. Rather, OX40 may mark highly suppressive Tregs in humans. OX40 Up-Regulation and Th1-Oriented Plasticity Occur in Distinct Treg Subpopulations To investigate whether Th1-like (mostly OX40–) and Th1-suppressing (mostly OX40+) subsets belonged to distinct Treg subpopulations, we performed an extensive multiparameter flow cytometric (FCM) analysis aimed at identifying a marker, or a set of markers, cosegregating with the two phenotypes, in Tregs freshly isolated from the different liver compartments. First, we subdivided Tregs into CD45RA+FOXP3low resting, CD45RA–FOXP3high activated (act-) Tregs, or CD45RA–FOXP3low nonsuppressive (non-) Tregs.27 We noticed that the subset of act-Tregs accumulated in TUM tissue in both nc and c pts, but was significantly higher in NT-LIV from c, compared to nc, pts (Fig. 4A). Both non-Tregs and act-Tregs, as well as the Tconv counterpart as a control, were further subdivided into two populations according to the high or low expression of Helios.20 This analysis revealed that Helios expression appeared to be mutually exclusive with respect to IFN-γ production (Fig. 4B), recalling what was observed for OX40 expression (see Fig. 3D). A relevant proportion of Helioshigh Tregs could be detected in the non-Treg gate, but Helioshigh Tregs were maximally enriched in the act-Treg gate (Supporting Fig. 3A). When cells were stratified into Helioshigh and Helioslow subsets, a clear cosegregation of multiple markers appeared. Indeed, within all the populations studied, the Helioshigh cells were enriched in OX40+ (PD-1+ or –), in T-bethighIFN-γ– and in proliferating (Ki67+) CD39+ cells (Fig. 4C). By contrast, the Helioslow subset was enriched in PD1+OX40–, T-bet+IFN-γ+, CD39–, and Ki67– cells (Fig. 4C). Some quantitative differences were observed among act-Tregs, non-Tregs, and Tconvs: (1) the Helioshigh act-Tregs contained the highest proportions of OX40+, T-bethighIFN-γ–, and CD39+Ki67+ cells and the lowest frequencies of PD1+OX40– cells, as compared with the Helioshigh counterparts in non-Tregs and Tconvs, and (2) the Helioshigh subgroup of both act-Tregs and non-Tregs contained the lowest fractions of T-bet+IFN-γ+, as compared with the Helioshigh Tconvs (Fig. 4C). When we compared samples from nc and c pts, we could observe that Helioshigh/low subsets showed similar frequency (Supporting Fig. 3A) and similar phenotype (in terms of OX40, PD-1, CD39, and Ki67 expression), but different plasticity: Indeed, Helioslow act-Tregs contained a higher Th1-like Treg frequency in nc versus c pts (Supporting Fig. 3B), suggesting that both quantitative (different act-Treg frequency; see Fig. 4A) and qualitative modulations might contribute to shaping the Treg pool in distinct microenvironments. To demonstrate an association between Helios expression and Treg commitment, we analyzed, in sorted Helioshigh or Helioslow act-Tregs or non-Tregs, the frequency of cells carrying a demethylated TSDR, critically controlling the specification of Treg lineage.3 We found that Helioshigh Tregs contained a significantly higher proportion of cells with demethylated TSDR, compared to Helioshigh Tregs, in both non-Tregs and act-Tregs subpopulations (Fig. 4D). We could not verify a higher TSDR demethylation in liver-infiltrating Helioshigh Tregs, because of the insufficient amounts of recovered cells required for this analysis. However, we could observe, in the bulk Treg pool obtained from NT-LIVc of a CHC pt, high levels of TSDR demethylation, in line with high Helioshigh frequency (Fig. 4D, right). In summary, our data demonstrate that OX40+ Th1-suppressing Tregs are mostly contained in Helioshigh, TSDR-demethylated, and act-Tregs, accumulating in cirrhosis and cancer, tempting us to hypothesize that OX40 may play a functional role in shaping the pool of activated, suppressive, and protumoral Tregs. Figure 4Open in figure viewerPowerPoint Helioshigh subset is enriched in OX40+ and Th1-suppressing Tregs. (A) Representative plots of CD45RA versus FOXP3 staining in gated Tregs in different specimens from CHC pts, showing subsets