Regulatory T cells and autoimmune hepatitis: What happens in the liver stays in the liver
Authors: Ye H. Oo, David H. Adams
Journal: Journal of Hepatology (2014)

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapiesJournal of HepatologyVol. 61Issue 5PreviewAutoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. Full-Text PDF Autoimmune hepatitis (AIH) is a classical autoimmune liver disease that has characteristic features of autoimmunity. It is more commonly seen in women, strongly associated with variants in the major histocompatibility complex region and usually responsive to immunosuppression. Although the aetiology is still unknown the development of the disease involves a loss of immunological self-tolerance to liver antigens resulting in a T cell mediated destruction of hepatocytes [[1]Tait B. Mackay I.R. Board P. Coggan M. Emery P. Eckardt G. HLA A1, B8, DR3 extended haplotypes in autoimmune chronic hepatitis.Gastroenterology. 1989; 97: 479-481Abstract Full Text PDF PubMed Google Scholar]. The majority of AIH patients require lifelong immunosuppression to maintain remission and the treatment for AIH still relies on corticosteroids and azathioprine, which were first introduced more than 25 years ago [[2]Cook G.C. Mulligan R. Sherlock S. Controlled prospective trial of corticosteroid therapy in active chronic hepatitis.QJM. 1971; 40: 159-185Crossref PubMed Scopus (430) Google Scholar]. In recent years it has been suggested that one of the factors that predisposes to autoimmune disease is an imbalance between effector and regulatory arms of the immune system [3Sakaguchi S. Sakaguchi N. Asano M. Itoh M. Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J Immunol. 1995; 155: 1151-1164PubMed Google Scholar, 4Putnam A.L. Safinia N. Medvec A. Laszkowska M. Wray M. Mintz M.A. et al.Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.Am J Transplant. 2013; 13: 3010-3020Crossref PubMed Scopus (200) Google Scholar]. Evidence of this comes not only from clinical studies but also from animal studies in which defects in immune regulation and a lack of functional regulatory T cells (Tregs) is associated with the development of autoimmunity including autoimmune hepatitis. However, it is less clear whether the defect in patients with AIH is due to a failure of regulatory cell recruitment or function, or whether regulatory cells are simply overwhelmed by dominant effector cell responses. The article by Taubert and colleagues in this edition of the Journal of Hepatology sheds more light on this debate. They conducted a correlative study using immunohistochemistry and flow cytometry to quantify the relative proportions of effector cells, regulatory T cells and B cells in the liver and blood of patients with type1 AIH. Consistent with other studies they reported that intrahepatic Tregs are enriched in untreated type1 AIH but that their numbers fall in response to therapy. The authors suggest that this effect of therapy might explain the high relapse rates seen after discontinuation of immunosuppression in AIH and speculate that therapies that increase or preserve intrahepatic immunoregulation might be better suited for the long-term control of AIH.Lymphocytes comprise around 25% of intrahepatic immune cells and include effector CD4 T cells, cytotoxic T cells, B cells, invariant iNKT, and MAIT cells [[5]Racanelli V. Rehermann B. The liver as an immunological organ.Hepatology. 2006; 43: S54-S62Crossref PubMed Scopus (861) Google Scholar]. Regulatory networks particularly intrahepatic CD4+CD25highCD127lowFOXP3+ regulatory T cells control the effector responses. The intrahepatic frequency of these cells varies from 1% to 5% of the lymphocyte population in most chronic liver diseases [[6]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] and is associated with a reversal of the CD4/CD8 ratio in the liver compared with blood [[7]Doherty D.G. O'Farrelly C. Innate and adaptive lymphoid cells in the human liver.Immunol Rev. 2000; 174: 5-20Crossref PubMed Scopus (307) Google Scholar]. In autoimmune hepatitis, CD4 T cells dominate in the early stage of the disease, particularly Th1 cells [[8]Lohr H.F. Schlaak J.F. Lohse A.W. Bocher W.O. Arenz M. Gerken G. et al.Autoreactive CD4+ LKM-specific and anticlonotypic T-cell responses in LKM-1 antibody-positive autoimmune hepatitis.Hepatology. 1996; 24: 1416-1421Crossref PubMed Google Scholar]. Subsequently, T helper cells recruit cytotoxic CD8 T cells leading to higher CD8 T cell frequencies and possibly more aggressive cytotoxic T cell mediated destruction of hepatocytes as the disease progresses as described by Taubert and colleagues [[9]Taubert R. Hardtke-Wolenski M. Noyan F. Wilms A. Baumann A.K. Schlue J. et al.Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.J Hepatol. 2014; 61: 1106-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar].Staining for the FOXP3 transcription factor, the master regulator of Treg differentiation and function has been widely used to quantify CD4 Tregs in tissues [[10]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 (6020) Google Scholar]. However, FOXP3 is also expressed transiently during T cell activation and in some other lymphocyte subsets, potentially reducing the reliability of single colour immunohistochemistry to quantify Tregs in hepatic inflammation [6Oo 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, 11Lan R.Y. Cheng C. Lian Z.X. Tsuneyama K. Yang G.X. Moritoki Y. et al.Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis.Hepatology. 2006; 43: 729-737Crossref PubMed Scopus (258) Google Scholar]. The current report partially overcomes this by using double immunohistochemistry. However, in the inflamed environment some Tregs may display lineage plasticity and express both FOXP3 and the Th1 transcription factor TBet [[12]Yang X.O. Nurieva R. Martinez G.J. Kang H.S. Chung Y. Pappu B.P. et al.Molecular antagonism and plasticity of regulatory and inflammatory T cell programs.Immunity. 2008; 29: 44-56Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar] and high local levels of TGF-β and retinoic acid may generate induced Tregs [13Carambia A. Freund B. Schwinge D. Heine M. Laschtowitz A. Huber S. et al.TGF-beta-dependent induction of CD4CD25Foxp3 Tregs by liver sinusoidal endothelial cells.J Hepatol. 2014; Google Scholar, 14Dunham R.M. Thapa M. Velazquez V.M. Elrod E.J. Denning T.L. Pulendran B. et al.Hepatic stellate cells preferentially induce Foxp3+ regulatory T cells by production of retinoic acid.J Immunol. 2013; 190: 2009-2016Crossref PubMed Scopus (98) Google Scholar]. A more detailed phenotypic analysis of freshly isolated intra-hepatic Tregs with multi-colour flow cytometry would answer these questions particularly if this includes the TSDR methylation status, which correlates closely with stability and function of thymic derived Tregs [[15]Polansky J.K. Kretschmer K. Freyer J. Floess S. Garbe A. Baron U. et al.DNA methylation controls Foxp3 gene expression.Eur J Immunol. 2008; 38: 1654-1663Crossref PubMed Scopus (614) Google Scholar].AIH flares are characterized by high frequencies of both Tregs and effector T cells (Teffs) in the inflamed liver [8Lohr H.F. Schlaak J.F. Lohse A.W. Bocher W.O. Arenz M. Gerken G. et al.Autoreactive CD4+ LKM-specific and anticlonotypic T-cell responses in LKM-1 antibody-positive autoimmune hepatitis.Hepatology. 1996; 24: 1416-1421Crossref PubMed Google Scholar, 9Taubert R. Hardtke-Wolenski M. Noyan F. Wilms A. Baumann A.K. Schlue J. et al.Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.J Hepatol. 2014; 61: 1106-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar] (Fig. 1). Now Taubert and colleagues show that patients reaching biochemical remission have higher intrahepatic Treg/Teff and Treg/B cell ratios compared to those who fail to respond. This suggests that Tregs are important in gaining and maintaining remission. The finding that intrahepatic Tregs decrease during therapy is novel and particularly interesting and could explain the high relapse rates after discontinuation of immunosuppression. The authors' data, showing that Tregs are more susceptible to particular forms of immunosuppression, suggest we should consider drugs that more specifically suppress effector cell responses. These observations are relevant for future therapy with infused Tregs and would suggest that Treg infusions will be most effective if used at induction or instead of maintaining immunosuppression. The current study reports that steroid and azathioprine therapy has a greater effect on intrahepatic Tregs compared with T effector cells [1Tait B. Mackay I.R. Board P. Coggan M. Emery P. Eckardt G. HLA A1, B8, DR3 extended haplotypes in autoimmune chronic hepatitis.Gastroenterology. 1989; 97: 479-481Abstract Full Text PDF PubMed Google Scholar, 9Taubert R. Hardtke-Wolenski M. Noyan F. Wilms A. Baumann A.K. Schlue J. et al.Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.J Hepatol. 2014; 61: 1106-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. An important immunosuppressive mechanism of action of prednisolone/glucocorticoid is the inhibition of IL2 gene expression and two other commonly used immunosuppressive drugs, cyclosporine and tacrolimus; also reduce IL-2 production [[16]Granelli-Piperno A. Nolan P. Inaba K. Steinman R.M. The effect of immunosuppressive agents on the induction of nuclear factors that bind to sites on the interleukin 2 promoter.J Exp Med. 1990; 172: 1869-1872Crossref PubMed Scopus (115) Google Scholar]. Tregs are highly dependent on IL-2 and this may explain the particular susceptibility of Tregs to treatment in the present study. Thus, it may be logical to use immunosuppressive drugs that preserve IL-2 mediated signalling in AIH.We have previously reported similar frequencies of intrahepatic effector and regulatory lymphocytes in liver tissue removed at transplantation from patients with autoimmune hepatitis [[6]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] but the present study is the first to describe this in steroid-naïve patients. The consistent frequencies of intrahepatic Tregs show that there is no failure of Treg recruitment to the liver in AIH, and suggest it is the balance of Tregs and effector cells that will dictate the outcome. It is thus important to understand the factors within the hepatic microenvironment that determine differential survival or activation of Tregs and effector cells (Fig. 1).A further observation in the present paper was that the frequency of intra-portal B cells correlates with levels of serum IgG suggesting that the elevated IgG levels that are characteristic of active AIH may be a consequence of local intrahepatic production. More research is required to understand the functional consequences of local B cell activation in the liver and the nature of the IgG secreting cells. The fact that IgG secretion falls on rituximab therapy despite the fact that CD20 is not expressed on plasma cells suggests that IgG autoantibodies may be secreted by memory B cells within inflammatory niches in tissue consistent with the findings of the present study [[17]Ferraro A.J. Drayson M.T. Savage C.O. MacLennan I.C. Levels of autoantibodies, unlike antibodies to all extrinsic antigen groups, fall following B cell depletion with Rituximab.Eur J Immunol. 2008; 38: 292-298Crossref PubMed Scopus (69) Google Scholar].Another important conclusion from the present study is that studies of Tregs in blood may not reflect what happens in tissues. Studies have reported different findings regarding the frequency and function of Tregs in the peripheral blood of patients with autoimmune hepatitis [18Longhi M.S. Hussain M.J. Mitry R.R. Arora S.K. Mieli-Vergani G. Vergani D. et al.Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis.J Immunol. 2006; 176: 4484-4491Crossref PubMed Scopus (277) Google Scholar, 19Peiseler M. Sebode M. Franke B. Wortmann F. Schwinge D. Quaas A. et al.FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency.J Hepatol. 2012; 57: 125-132Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 20Ferri S. Longhi M.S. De Molo C. Lalanne C. Muratori P. Granito A. et al.A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis.Hepatology. 2010; 52: 999-1007Crossref PubMed Scopus (142) Google Scholar], which emphasizes the importance of studying intrahepatic Tregs and not over-interpreting findings from blood. Finally, analysis of the makeup of hepatic infiltrates in AIH may provide useful to predict outcome and response to therapy as well as helping to predict which patients can have immunosuppression withdrawn without a high risk of relapse.In conclusion, the present study provides more evidence on the important role of intrahepatic Tregs vs. effector T cells and B cells in autoimmune hepatitis. A detailed study of Tregs in the liver tissues and how inflammation effects their local survival and function will provide closer understanding of Tregs in autoimmune hepatitis (Fig. 1). Functional rather than phenotypic studies are required to gain the necessary insights into the pathogenesis but they are difficult to perform. Correlation of immunophenotypic data with clinical data is helpful as suggested in the current study. Immunoregulatory mechanisms in the human liver are more complicated than previously thought and understanding these aspects will allow us to develop more specific therapy in AIH that gets away from blanket immunosuppression.Financial supportYe Htun Oo was funded by the Medical Research Council Intermediate fellowship programme.Conflict of interestThe authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Autoimmune hepatitis (AIH) is a classical autoimmune liver disease that has characteristic features of autoimmunity. It is more commonly seen in women, strongly associated with variants in the major histocompatibility complex region and usually responsive to immunosuppression. Although the aetiology is still unknown the development of the disease involves a loss of immunological self-tolerance to liver antigens resulting in a T cell mediated destruction of hepatocytes [[1]Tait B. Mackay I.R. Board P. Coggan M. Emery P. Eckardt G. HLA A1, B8, DR3 extended haplotypes in autoimmune chronic hepatitis.Gastroenterology. 1989; 97: 479-481Abstract Full Text PDF PubMed Google Scholar]. The majority of AIH patients require lifelong immunosuppression to maintain remission and the treatment for AIH still relies on corticosteroids and azathioprine, which were first introduced more than 25 years ago [[2]Cook G.C. Mulligan R. Sherlock S. Controlled prospective trial of corticosteroid therapy in active chronic hepatitis.QJM. 1971; 40: 159-185Crossref PubMed Scopus (430) Google Scholar]. In recent years it has been suggested that one of the factors that predisposes to autoimmune disease is an imbalance between effector and regulatory arms of the immune system [3Sakaguchi S. Sakaguchi N. Asano M. Itoh M. Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J Immunol. 1995; 155: 1151-1164PubMed Google Scholar, 4Putnam A.L. Safinia N. Medvec A. Laszkowska M. Wray M. Mintz M.A. et al.Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.Am J Transplant. 2013; 13: 3010-3020Crossref PubMed Scopus (200) Google Scholar]. Evidence of this comes not only from clinical studies but also from animal studies in which defects in immune regulation and a lack of functional regulatory T cells (Tregs) is associated with the development of autoimmunity including autoimmune hepatitis. However, it is less clear whether the defect in patients with AIH is due to a failure of regulatory cell recruitment or function, or whether regulatory cells are simply overwhelmed by dominant effector cell responses. The article by Taubert and colleagues in this edition of the Journal of Hepatology sheds more light on this debate. They conducted a correlative study using immunohistochemistry and flow cytometry to quantify the relative proportions of effector cells, regulatory T cells and B cells in the liver and blood of patients with type1 AIH. Consistent with other studies they reported that intrahepatic Tregs are enriched in untreated type1 AIH but that their numbers fall in response to therapy. The authors suggest that this effect of therapy might explain the high relapse rates seen after discontinuation of immunosuppression in AIH and speculate that therapies that increase or preserve intrahepatic immunoregulation might be better suited for the long-term control of AIH. Lymphocytes comprise around 25% of intrahepatic immune cells and include effector CD4 T cells, cytotoxic T cells, B cells, invariant iNKT, and MAIT cells [[5]Racanelli V. Rehermann B. The liver as an immunological organ.Hepatology. 2006; 43: S54-S62Crossref PubMed Scopus (861) Google Scholar]. Regulatory networks particularly intrahepatic CD4+CD25highCD127lowFOXP3+ regulatory T cells control the effector responses. The intrahepatic frequency of these cells varies from 1% to 5% of the lymphocyte population in most chronic liver diseases [[6]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] and is associated with a reversal of the CD4/CD8 ratio in the liver compared with blood [[7]Doherty D.G. O'Farrelly C. Innate and adaptive lymphoid cells in the human liver.Immunol Rev. 2000; 174: 5-20Crossref PubMed Scopus (307) Google Scholar]. In autoimmune hepatitis, CD4 T cells dominate in the early stage of the disease, particularly Th1 cells [[8]Lohr H.F. Schlaak J.F. Lohse A.W. Bocher W.O. Arenz M. Gerken G. et al.Autoreactive CD4+ LKM-specific and anticlonotypic T-cell responses in LKM-1 antibody-positive autoimmune hepatitis.Hepatology. 1996; 24: 1416-1421Crossref PubMed Google Scholar]. Subsequently, T helper cells recruit cytotoxic CD8 T cells leading to higher CD8 T cell frequencies and possibly more aggressive cytotoxic T cell mediated destruction of hepatocytes as the disease progresses as described by Taubert and colleagues [[9]Taubert R. Hardtke-Wolenski M. Noyan F. Wilms A. Baumann A.K. Schlue J. et al.Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.J Hepatol. 2014; 61: 1106-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Staining for the FOXP3 transcription factor, the master regulator of Treg differentiation and function has been widely used to quantify CD4 Tregs in tissues [[10]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 (6020) Google Scholar]. However, FOXP3 is also expressed transiently during T cell activation and in some other lymphocyte subsets, potentially reducing the reliability of single colour immunohistochemistry to quantify Tregs in hepatic inflammation [6Oo 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, 11Lan R.Y. Cheng C. Lian Z.X. Tsuneyama K. Yang G.X. Moritoki Y. et al.Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis.Hepatology. 2006; 43: 729-737Crossref PubMed Scopus (258) Google Scholar]. The current report partially overcomes this by using double immunohistochemistry. However, in the inflamed environment some Tregs may display lineage plasticity and express both FOXP3 and the Th1 transcription factor TBet [[12]Yang X.O. Nurieva R. Martinez G.J. Kang H.S. Chung Y. Pappu B.P. et al.Molecular antagonism and plasticity of regulatory and inflammatory T cell programs.Immunity. 2008; 29: 44-56Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar] and high local levels of TGF-β and retinoic acid may generate induced Tregs [13Carambia A. Freund B. Schwinge D. Heine M. Laschtowitz A. Huber S. et al.TGF-beta-dependent induction of CD4CD25Foxp3 Tregs by liver sinusoidal endothelial cells.J Hepatol. 2014; Google Scholar, 14Dunham R.M. Thapa M. Velazquez V.M. Elrod E.J. Denning T.L. Pulendran B. et al.Hepatic stellate cells preferentially induce Foxp3+ regulatory T cells by production of retinoic acid.J Immunol. 2013; 190: 2009-2016Crossref PubMed Scopus (98) Google Scholar]. A more detailed phenotypic analysis of freshly isolated intra-hepatic Tregs with multi-colour flow cytometry would answer these questions particularly if this includes the TSDR methylation status, which correlates closely with stability and function of thymic derived Tregs [[15]Polansky J.K. Kretschmer K. Freyer J. Floess S. Garbe A. Baron U. et al.DNA methylation controls Foxp3 gene expression.Eur J Immunol. 2008; 38: 1654-1663Crossref PubMed Scopus (614) Google Scholar]. AIH flares are characterized by high frequencies of both Tregs and effector T cells (Teffs) in the inflamed liver [8Lohr H.F. Schlaak J.F. Lohse A.W. Bocher W.O. Arenz M. Gerken G. et al.Autoreactive CD4+ LKM-specific and anticlonotypic T-cell responses in LKM-1 antibody-positive autoimmune hepatitis.Hepatology. 1996; 24: 1416-1421Crossref PubMed Google Scholar, 9Taubert R. Hardtke-Wolenski M. Noyan F. Wilms A. Baumann A.K. Schlue J. et al.Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.J Hepatol. 2014; 61: 1106-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar] (Fig. 1). Now Taubert and colleagues show that patients reaching biochemical remission have higher intrahepatic Treg/Teff and Treg/B cell ratios compared to those who fail to respond. This suggests that Tregs are important in gaining and maintaining remission. The finding that intrahepatic Tregs decrease during therapy is novel and particularly interesting and could explain the high relapse rates after discontinuation of immunosuppression. The authors' data, showing that Tregs are more susceptible to particular forms of immunosuppression, suggest we should consider drugs that more specifically suppress effector cell responses. These observations are relevant for future therapy with infused Tregs and would suggest that Treg infusions will be most effective if used at induction or instead of maintaining immunosuppression. The current study reports that steroid and azathioprine therapy has a greater effect on intrahepatic Tregs compared with T effector cells [1Tait B. Mackay I.R. Board P. Coggan M. Emery P. Eckardt G. HLA A1, B8, DR3 extended haplotypes in autoimmune chronic hepatitis.Gastroenterology. 1989; 97: 479-481Abstract Full Text PDF PubMed Google Scholar, 9Taubert R. Hardtke-Wolenski M. Noyan F. Wilms A. Baumann A.K. Schlue J. et al.Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.J Hepatol. 2014; 61: 1106-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. An important immunosuppressive mechanism of action of prednisolone/glucocorticoid is the inhibition of IL2 gene expression and two other commonly used immunosuppressive drugs, cyclosporine and tacrolimus; also reduce IL-2 production [[16]Granelli-Piperno A. Nolan P. Inaba K. Steinman R.M. The effect of immunosuppressive agents on the induction of nuclear factors that bind to sites on the interleukin 2 promoter.J Exp Med. 1990; 172: 1869-1872Crossref PubMed Scopus (115) Google Scholar]. Tregs are highly dependent on IL-2 and this may explain the particular susceptibility of Tregs to treatment in the present study. Thus, it may be logical to use immunosuppressive drugs that preserve IL-2 mediated signalling in AIH. We have previously reported similar frequencies of intrahepatic effector and regulatory lymphocytes in liver tissue removed at transplantation from patients with autoimmune hepatitis [[6]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] but the present study is the first to describe this in steroid-naïve patients. The consistent frequencies of intrahepatic Tregs show that there is no failure of Treg recruitment to the liver in AIH, and suggest it is the balance of Tregs and effector cells that will dictate the outcome. It is thus important to understand the factors within the hepatic microenvironment that determine differential survival or activation of Tregs and effector cells (Fig. 1). A further observation in the present paper was that the frequency of intra-portal B cells correlates with levels of serum IgG suggesting that the elevated IgG levels that are characteristic of active AIH may be a consequence of local intrahepatic production. More research is required to understand the functional consequences of local B cell activation in the liver and the nature of the IgG secreting cells. The fact that IgG secretion falls on rituximab therapy despite the fact that CD20 is not expressed on plasma cells suggests that IgG autoantibodies may be secreted by memory B cells within inflammatory niches in tissue consistent with the findings of the present study [[17]Ferraro A.J. Drayson M.T. Savage C.O. MacLennan I.C. Levels of autoantibodies, unlike antibodies to all extrinsic antigen groups, fall following B cell depletion with Rituximab.Eur J Immunol. 2008; 38: 292-298Crossref PubMed Scopus (69) Google Scholar]. Another important conclusion from the present study is that studies of Tregs in blood may not reflect what happens in tissues. Studies have reported different findings regarding the frequency and function of Tregs in the peripheral blood of patients with autoimmune hepatitis [18Longhi M.S. Hussain M.J. Mitry R.R. Arora S.K. Mieli-Vergani G. Vergani D. et al.Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis.J Immunol. 2006; 176: 4484-4491Crossref PubMed Scopus (277) Google Scholar, 19Peiseler M. Sebode M. Franke B. Wortmann F. Schwinge D. Quaas A. et al.FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency.J Hepatol. 2012; 57: 125-132Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 20Ferri S. Longhi M.S. De Molo C. Lalanne C. Muratori P. Granito A. et al.A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis.Hepatology. 2010; 52: 999-1007Crossref PubMed Scopus (142) Google Scholar], which emphasizes the importance of studying intrahepatic Tregs and not over-interpreting findings from blood. Finally, analysis of the makeup of hepatic infiltrates in AIH may provide useful to predict outcome and response to therapy as well as helping to predict which patients can have immunosuppression withdrawn without a high risk of relapse. In conclusion, the present study provides more evidence on the important role of intrahepatic Tregs vs. effector T cells and B cells in autoimmune hepatitis. A detailed study of Tregs in the liver tissues and how inflammation effects their local survival and function will provide closer understanding of Tregs in autoimmune hepatitis (Fig. 1). Functional rather than phenotypic studies are required to gain the necessary insights into the pathogenesis but they are difficult to perform. Correlation of immunophenotypic data with clinical data is helpful as suggested in the current study. Immunoregulatory mechanisms in the human liver are more complicated than previously thought and understanding these aspects will allow us to develop more specific therapy in AIH that gets away from blanket immunosuppression. Financial supportYe Htun Oo was funded by the Medical Research Council Intermediate fellowship programme. Ye Htun Oo was funded by the Medical Research Council Intermediate fellowship programme. Conflict of interestThe authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. We thank Medical Research Council for Clinician Scientist funding for Dr Ye Htun Oo. We also thank National Institute for Health Research (NIHR- UK) for funding to carry out translational liver research in Birmingham, UK.