Regulatory T cells and autoimmune hepatitis: Defective cells or a hostile environment?

FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequencyJournal of HepatologyVol. 57Issue 1PreviewThe pathogenesis of autoimmune hepatitis (AIH) is not understood, but it was suggested that AIH may be related to a numerical or functional impairment of CD4+CD25+FOXP3+ regulatory T cells (Treg), which are important mediators of immune tolerance to self-antigens. However, the role of Treg in AIH is not clear, since earlier studies reporting Treg impairment had used only CD25 as marker that cannot unambiguously distinguish Treg from activated effector T cells. Full-Text PDF Reply to: “Regulatory T cells in autoimmune hepatitis”Journal of HepatologyVol. 57Issue 4PreviewWe read with interest the comment by Longhi et al. on our paper on Tregs in autoimmune hepatitis (AIH) [1] and the accompanying editorial by Oo and Adams [2]. The discrepant results by Longhi et al. and us on Treg function in autoimmune hepatitis (AIH) may originate from the use of different methods to purify and assess the suppressor activity of Tregs [3,4]. Therefore, we will reply to the criticism by comparing the methodologies used by the Kings College group and by us, and explain why we chose the methodology as we did. Full-Text PDF Open AccessRegulatory T cells in autoimmune hepatitisJournal of HepatologyVol. 57Issue 4PreviewWe read with interest the paper by Peiseler et al., and the accompanying editorial by Oo and Adams, reporting that in patients with autoimmune hepatitis (AIH), regulatory T cells (T-regs) are ‘fully functional and not reduced in frequency’ [1]. These data contrast with our findings of T-reg impairment in AIH, especially during active disease [2–5]. This discrepancy is attributed by the authors of the paper and the editorial to their use of an ‘improved’ methodology to define ‘true’ T-regs, i.e. CD4+CD25highCD127low cells. Full-Text PDF Open Access Autoimmunity occurs when genetically predisposed individuals are exposed to environmental factors that trigger immune responses to self-antigens. The immune system protects the host by recognising and removing invading pathogens and damaged cells and to do this efficiently it must be able to discriminate between infected or damaged cells and healthy self-tissue. This tolerance of self involves both central, thymic mechanisms and peripheral pathways involving regulatory T cells (Treg) (Fig. 1) [[1]Sakaguchi S. Yamaguchi T. Nomura T. Ono M. Regulatory T cells and immune tolerance.Cell. 2008; 133: 775-787Abstract Full Text Full Text PDF PubMed Scopus (3578) Google Scholar]. Regulatory T cells are a subpopulation of T cells which downregulate immune responses, maintain tolerance to self-antigens and prevent the development of autoimmunity. CD4 Treg, defined by high levels of CD25, low levels of CD127, and expression of the transcription factor FOXP3, comprise 1–5% of circulating T cells in humans [[2]Liu W. Putnam A.L. Xu-Yu Z. Szot G.L. Lee M.R. Zhu S. et al.CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.J Exp Med. 2006; 203: 1701-1711Crossref PubMed Scopus (2044) Google Scholar]. FOXP3, which regulates their development, is required for suppressive function and mice and human lacking functional FOXP3 develop multiorgan autoimmunity which in humans takes the form of the IPEX syndrome [[3]Fontenot J.D. Gavin M.A. Rudensky A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (5988) Google Scholar]. It is thus logical to look for defective Treg function in autoimmune hepatitis, an archetypal autoimmune disease [[4]Czaja A.J. Manns M.P. Advances in the diagnosis, pathogenesis, and management of autoimmune hepatitis.Gastroenterology. 2010; 139: e54Google Scholar]. Although previous studies reported reduced numbers and defective Treg function in AIH [5Longhi M.S. Ma Y. Bogdanos D.P. Cheeseman P. Mieli-Vergani G. Vergani D. Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease.J Hepatol. 2004; 41: 31-37Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 6Longhi M.S. Hussain M.J. Kwok W.W. Mieli-Vergani G. Ma Y. Vergani D. Autoantigen-specific regulatory T cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis.Hepatology. 2011; 53: 536-547Crossref PubMed Scopus (90) Google Scholar, 7Longhi M.S. Hussain M.J. Bogdanos D.P. Quaglia A. Mieli-Vergani G. Ma Y. et al.Cytochrome P450IID6-specific CD8 T cell immune responses mirror disease activity in autoimmune hepatitis type 2.Hepatology. 2007; 46: 472-484Crossref PubMed Scopus (105) Google Scholar], Peiseler et al. now report normal frequencies and function of Treg in patients with type 1 AIH; indeed they found higher Treg frequencies in blood and liver tissue during active disease compared with remission. How can these different reports be reconciled? One explanation is the use of better Treg markers in the current study. Earlier studies used CD25 expression to define Treg but CD25 is also expressed on effector CD4 T cells. Peiseler et al. used low levels of CD127 and FOXP3 demethylation to accurately distinguish Treg from effector T cells. In humans, FOXP3 is transiently upregulated during T cell activation but constitutive expression, which is necessary for Treg function, requires demethylation of CpG dinucleotides at the FOXP3 locus providing an epigenetic marker to discriminate between Treg and effector T cells [[8]Baron U. Floess S. Wieczorek G. Baumann K. Grutzkau A. Dong J. et al.DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells.Eur J Immunol. 2007; 37: 2378-2389Crossref PubMed Scopus (534) Google Scholar]. They also quantified Treg within the liver and confirmed previous findings that intrahepatic Treg frequencies mirror those of effector T cells [[9]Oo Y.H. Weston C.J. Lalor P.F. Curbishley S.M. Withers D.R. Reynolds G.M. et al.Distinct roles for CCR4 and CXCR3 in the recruitment and positioning of regulatory T cells in the inflamed human liver.J Immunol. 2010; 184: 2886-2898Crossref PubMed Scopus (171) Google Scholar]. Thus, on balance, it seems unlikely that a reduced frequency of Treg underlies immune escape in AIH. If Treg are dysfunctional, this could explain failed immune suppression in the presence of normal numbers. Unlike some previous studies, the current paper failed to detect functional defects in Treg during active AIH or during remission. However, a recent study suggests that a combination of reduced Tim-3 levels on effector T cells and Gal9 on Treg renders effector cells resistant to Treg control in AIH [[10]Liberal R. Grant C.R. Holder B. Ma Y. Mieli-Vergani G. Vergani D. et al.The impaired immune regulation of autoimmune hepatitis is linked to a defective Galectin-9/Tim-3 pathway.Hepatology. 2012; (http://dx.doi.org/10.1002/hep.25682)PubMed Google Scholar] implicating defective T cell activation as well as Treg in the failed immune regulation. This draws parallels with studies reporting defective T cell regulation in chronic viral hepatitis [11Golden-Mason L. Palmer B.E. Kassam N. Townshend-Bulson L. Livingston S. McMahon B.J. et al.Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells.J Virol. 2009; 83: 9122-9130Crossref PubMed Scopus (358) Google Scholar, 12Rushbrook S.M. Ward S.M. Unitt E. Vowler S.L. Lucas M. Klenerman P. et al.Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.J Virol. 2005; 79: 7852-7859Crossref PubMed Scopus (241) Google Scholar]. Thus, conventional assays of Treg function may be too simplistic to detect specific defects. An additional confounding factor may arise from studying Treg from blood rather than within tissues where they operate. Most published studies show increased hepatic infiltration of Treg in AIH and the relevant cells may have been recruited from the blood into tissue in response to inflammation. The complex cellular and cytokine environment of the inflamed liver may have profound effects on Treg differentiation, stability, and function as well as altering the susceptibility of effector T cells to suppression. In murine models, Treg appear to be functional and stable in the inflamed environment [[13]Rubtsov Y.P. Niec R.E. Josefowicz S. Li L. Darce J. Mathis D. et al.Stability of the regulatory T cell lineage in vivo.Science. 2010; 329: 1667-1671Crossref PubMed Scopus (516) Google Scholar] but human hepatic Treg show low levels of phosphorylated STAT-5 consistent with impaired IL-2 receptor signalling [[9]Oo Y.H. Weston C.J. Lalor P.F. Curbishley S.M. Withers D.R. Reynolds G.M. et al.Distinct roles for CCR4 and CXCR3 in the recruitment and positioning of regulatory T cells in the inflamed human liver.J Immunol. 2010; 184: 2886-2898Crossref PubMed Scopus (171) Google Scholar]. This might be a consequence of reduced secretion or increased consumption of IL-2 within the inflamed liver. Furthermore, Treg in tissue interact with other cells including dendritic and stromal cells which may affect their survival and function and together with local cytokines could reprogramme Treg to express either Tbet and a Th1 phenotype [[14]Koch M.A. Tucker-Heard G. Perdue N.R. Killebrew J.R. Urdahl K.B. Campbell D.J. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation.Nat Immunol. 2009; 10: 595-602Crossref PubMed Scopus (900) Google Scholar] or RORc and a Th-17 phenotype [[15]Voo K.S. Wang Y.H. Santori F.R. Boggiano C. Arima K. Bover L. et al.Identification of IL-17-producing FOXP3+ regulatory T cells in humans.Proc Natl Acad Sci USA. 2009; 106: 4793-4798Crossref PubMed Scopus (554) Google Scholar]. Thus, understanding the role of Treg in disease depends on better methods to study their interactions and functions within the tissue microenvironment. The normal liver contains specialised antigen-presenting cells, including hepatic dendritic cells and endothelial cells, that promote Treg generation and the presence of TGF-β, IL-10 and retinoic acid further promote the generation and survival of Treg [[16]Thomson A.W. Knolle P.A. Antigen-presenting cell function in the tolerogenic liver environment.Nat Rev Immunol. 2010; 10: 753-766Crossref PubMed Scopus (538) Google Scholar]. Such a network may be important to maintain immune homeostasis in the face of gut-derived antigens. This tolerogenic environment is disrupted in hepatitis when the innate immune response to local tissue injury and inflammation is followed by the recruitment of effector and regulatory lymphocytes which both use the chemokine receptor CXCR3 to respond to interferon-inducible chemokine ligands at sites of active hepatitis and enter the inflamed liver [17Santodomingo-Garzon T. Han J. Le T. Yang Y. Swain M.G. Natural killer T cells regulate the homing of chemokine CXC receptor 3-positive regulatory T cells to the liver in mice.Hepatology. 2009; 49: 1267-1276Crossref PubMed Scopus (78) Google Scholar, 9Oo Y.H. Weston C.J. Lalor P.F. Curbishley S.M. Withers D.R. Reynolds G.M. et al.Distinct roles for CCR4 and CXCR3 in the recruitment and positioning of regulatory T cells in the inflamed human liver.J Immunol. 2010; 184: 2886-2898Crossref PubMed Scopus (171) Google Scholar, 18Curbishley S.M. Eksteen B. Gladue R.P. Lalor P. Adams D.H. CXCR 3 activation promotes lymphocyte transendothelial migration across human hepatic endothelium under fluid flow.Am J Pathol. 2005; 167: 887-899Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. In type 2 AIH, Treg recognise the same autoantigen as effector T cells although the autoantigens in type 1 AIH remain unknown [[6]Longhi M.S. Hussain M.J. Kwok W.W. Mieli-Vergani G. Ma Y. Vergani D. Autoantigen-specific regulatory T cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis.Hepatology. 2011; 53: 536-547Crossref PubMed Scopus (90) Google Scholar]. It is possible that any defects could be confined to the antigen-specific Treg population within the liver and thus not readily detectable by measuring global Treg function in blood. Whether local suppression requires the presence of antigen-specific Treg or can be mediated by non-specific mechanisms remains unknown but is critical for the success of Treg therapy. Another confounding factor is the use of immunosuppressive therapy to treat AIH. In the present study, patients who had untreated active disease had higher frequencies of Treg and effector T cells compared with those with active disease on treatment and those in remission, demonstrating the importance of assessing Treg in untreated patients wherever possible. What are the therapeutic implications of these findings? Clinical trials show efficacy of Treg infusions in clinical GVHD after allogeneic stem cell transplantation [[19]Di Ianni M. Falzetti F. Carotti A. Terenzi A. Castellino F. Bonifacio E. et al.Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.Blood. 2011; 117: 3921-3928Crossref PubMed Scopus (799) Google Scholar] and using them to restore immune homeostasis in AIH without the need for toxic immunosuppressive therapy is an attractive option. The finding that functional Treg in AIH are present in blood and liver suggests that therapeutic success will depend on more than simply replacing a defective compartment. There is a need to shift the balance from damage towards resolution of inflammation emphasising the importance of understanding the effects of the local inflammatory microenvironment. Treatment will fail if adoptively transferred Treg are inhibited or pushed into an effector differentiation pathway by local factors in the liver. The use of drugs that maintain Treg differentiation including azacitidine which modulates FOXP3 DNA methylation and the immunosuppressive mTOR inhibitor sirolimus may facilitate Treg stability in vivo [[20]Goodyear O.C. Dennis M. Jilani N.Y. Loke J. Siddique S. Ryan G. et al.Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia.Blood. 2012; 119: 3361-3369Crossref PubMed Scopus (287) Google Scholar]. Another important factor is whether therapeutic Treg need to be antigen-specific; if so, such an approach is possible in type 2 AIH where the antigen is known but not in type 1 AIH or other autoimmune liver diseases where the antigen is unknown. Here, one has to hope that polyclonal Treg will be effective. Finally, we need to know how to deliver Treg and whether we want them to exert their action in lymphoid tissue or within the liver itself. Although there are challenges, the recent study showing that IL-2 infusions can mediate immunosuppressive activity associated with increased circulating Treg in HCV-related vasculitis provides encouragement to pursue Treg therapy in autoimmune liver disease [[21]Saadoun D. Rosenzwajg M. Joly F. Six A. Carrat F. Thibault V. et al.Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis.N Engl J Med. 2011; 365: 2067-2077Crossref PubMed Scopus (568) Google Scholar]. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.