From Bench to Bedside in Hepatology Dig Dis 2014;32:502–506 DOI: 10.1159/000360493
Future Perspective: Immunomodulatory Therapy for Autoimmune Hepatitis Marcial Sebode Ansgar W. Lohse I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Abstract In the last two decades, more and more light has been shed on the immunologic pathogenesis of autoimmune liver diseases, notably autoimmune hepatitis (AIH). An immunologic dysbalance with proinflammatory immune responses dominating over hepatic tolerance seems to be part of AIH pathogenesis. In detail, an impairment of regulatory T cells (Treg) is suspected. If this holds true and reduced Treg numbers or their function are pathogenic for AIH, this offers the option of cellular or immunomodulatory therapy. However, the exact immunological role of Treg in AIH still needs to be clarified before cellular therapy is promising for patients. © 2014 S. Karger AG, Basel
Introduction
The liver is continuously exposed to antigens provided by portal venous blood flow. To avoid overwhelming inflammatory responses, a network of hepatic immune cells preserves the balance between tolerance and defense against infectious diseases (fig. 1). The hepatic immune microenvironment turns the liver into a tolerant organ © 2014 S. Karger AG, Basel 0257–2753/14/0325–0502$39.50/0 E-Mail [email protected] www.karger.com/ddi
[1]. This is evident, for example, from the well-known observation that transplanted livers are better tolerated than other organs [2]. Hepatic tolerance also impacts on other inflamed organs: in the mouse model of multiple sclerosis the clinical phenotype was ameliorated once the neuronal autoantigen was expressed in the liver [3]. In the state of autoimmune liver disease a dysbalance has been suggested which may account for an autoimmune reaction against hepatocytes in autoimmune hepatitis (AIH) or against the biliary tree in primary biliary cirrhosis and in primary sclerosing cholangitis. Research has mainly concentrated on the impaired immune regulation in AIH. Therefore, this article will focus on the disrupted immune homeostasis in AIH and discuss the option of immunomodulatory treatment.
Immunological Dysbalance in AIH
AIH is a chronic inflammatory disease of the liver that is characterized by elevated transaminases and raised immunoglobulins, notably of immunoglobulin G, as well as autoantibodies [reviewed in 4, 5]. The autoimmune etiology of AIH is widely accepted. The disease occurs predominantly in women and in most cases remission can be induced by immunosuppressive treatment. However, the reason why hepatic tolerance is broken and which immune reactions are specific for AIH is not yet fully understood. Prof. Dr. A.W. Lohse I. Department of Medicine University Medical Center Hamburg-Eppendorf Martinistrasse 52, DE–20246 Hamburg (Germany) E-Mail alohse @ uke.de
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Key Words Autoimmune hepatitis · Regulatory T cells · Immune regulation · Cellular therapy
Fig. 1. Immune homeostasis in AIH. Self-antigens are presented to the immune system by hepatocytes or other antigen-presenting cells (APC). In AIH, effector T cells including CD4+, CD8+ or Th17 cells recognize these antigens and promote liver inflammation. Treg that are probably antigen-specific normally limit hepatic inflammation.
Effector cells mediate the inflammation of autoimmune liver disease. First CD4+ and CD8+ T cells were recognized to be responsible for hepatocellular damage. A CD4+ or Th1 immune response was favored in two different mouse models of AIH [14, 15]. In another model and in peripheral blood of AIH patients, CD8+ T cells were the main effector cell type [16, 17]. Th17 cells, a Thelper cell subtype that is characterized by proinflammatory interleukin (IL)-17, appear to be involved in the pathogenesis of autoimmune diseases, e.g. multiple sclerosis or rheumatoid arthritis [18, 19]. Even in autoimmune liver diseases there seems to be a role for IL-17-producing cells [20–22]. If IL-17 plays a major role in autoimmune liver disease, a neutralizing IL-17 antibody could offer specific blockade of liver inflammation [23] and avoid general immunosuppression by standard therapy. However, an unsolved question is whether hepatic IL17-producing cells are pathogenic for autoimmune liver diseases or whether they solely represent an inflammatory bystander reaction [24]. Besides CD8+, Th1 and Th17 cells, innate immune cells such as NK cells or unconventional T cells like γδ T and NKT cells could play a role in disrupted immune homeostasis of AIH, but data on these cell types is limited.
It is assumed that tolerance to liver antigens is lost in AIH, which leads to the activation of autoreactive T cells. Specific T cells have been identified to react with CYP2D6 or SepSecS autoantigens [6, 7]. Still, except for the abovementioned autoantigens in type 2 or anti-SLA/LP (soluble liver antigen/liver pancreas antigen)-positive type 1 AIH, the main autoantigens of AIH remain elusive. Antigen presentation is mediated by human leukocyte antigens (HLA). An immunogenic predisposition is reflected by the association of AIH with HLA subtypes, e.g. type 1 AIH is preferentially associated with HLA-DRB1*0301 and HLA-DRB1*0401 [8]. HLA subtypes have an influence on the therapeutic response and clinical course [9]. As an important part of the hepatic microenvironment, antigen-presenting cells can shape immune reactions [1, 10]. Hepatic antigen-presenting cells comprise hepatocytes, Kupffer cells, dendritic cells, stellate cells and liver sinusoidal endothelial cells. Antigen-presenting cells like liver sinusoidal endothelial cells could play a role in AIH by modulating T-effector cell responses or induction of tolerance [11–13].
Apart from identifying the pathogenic proinflammatory responses, the impaired anti-inflammatory immunoregulation in AIH has been thoroughly investigated. Regulatory T cells (Treg) are important mediators of immune tolerance and their impairment seems to be involved in autoimmune diseases [25]. Treg are capable of suppressing the effector functions of various cell types. Hints for a role of Treg in AIH come from a mouse model of spontaneous liver inflammation which combined the loss of Treg with the disruption of immunoregulatory signalling by the molecule ‘PD-1’ (programmed cell death-1) [26]. Several studies from King’s College London propose a global impairment of Treg function and numbers in pediatric AIH patients [reviewed in 27]. In a very recent publication, Grant et al. [28] showed that Treg from AIH patients fail to suppress proinflammatory IL17 by CD4+ T cells. Still, the role of Treg in the pathogenesis of AIH is not entirely clear. One reason lies in the fact that specific definition of Treg by intra- and extracellular markers is challenging. In early studies, Treg were defined as CD4+CD25+ T cells. In the following years it became
Future Perspective: Immunomodulatory Therapy for Autoimmune Hepatitis
Dig Dis 2014;32:502–506 DOI: 10.1159/000360493
503
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Regulatory T Cells in AIH
Immunomodulatory Therapy for AIH
Standard immunosuppressive treatment for AIH consists of corticosteroids in combination with azathioprine. Thereby, remission is induced in most patients. This suggests that T-effector cells are limited by immunosuppression. Whether azathioprine and prednisolone play an auxiliary immunomodulatory role in AIH is unclear. This has been proposed for prednisolone in other autoimmune diseases like myasthenia gravis [34]. However, the pathogenic immune reaction of AIH is not terminated by classical immunosuppressive therapy. That is why recurrent courses of AIH can occur after tapering immunosuppression. A more selective therapy for AIH is highly desirable. Immunomodulatory approaches for AIH have mainly focused on the Treg population. In order to support tolerance in the liver, efforts to stabilize or even enlarge Treg have been undertaken. The mTOR inhibitor rapamycin or the vitamin A metabolite retinoic acid can promote Treg numbers [35, 36]. Another agent supporting Treg recovery is IL-2 [37]. In pediatric patients with primary sclerosing cholangitis and inflammatory bowel disease, the oral antibiotic vancomycin had immunomodulatory effects and resulted in increased peripheral Treg numbers and normalization of liver function tests [38]. Secondline therapy for AIH such as anti-TNF-α treatment could 504
Dig Dis 2014;32:502–506 DOI: 10.1159/000360493
also imply immunomodulation [39]. It has been shown that anti-TNF-α treatment reversed the reduction of peripheral Treg numbers and their increased apoptosis in inflammatory bowel disease [40]. To create large numbers of Treg, the best approach seems to be the adoptive transfer of ex vivo expanded Treg. In a mouse model of type 2 AIH, transferred antigen-specific Treg were able to restore peripheral tolerance [41]. This was confirmed in humans by Longhi et al. [42] who generated CYP2D6-specific Treg that efficiently suppressed in vitro autoreactive CD4+ T cells derived from type 2 AIH patients. In general, mouse models have shown that antigen-specific Treg are more effective in suppressing autoimmune diseases than polyclonal Treg [reviewed in 43]. Unfortunately, in most cases of type 1 AIH the autoantigen is unknown and therefore only polyclonal Treg are available for adoptive transfer. It is uncertain whether in human autoimmune diseases polyclonal Treg are capable of suppressing effector cells. Several technical problems still need to be overcome before adoptive transfer of Treg becomes applicable for clinical studies in AIH [reviewed in 44]. One general concern is safety since the administration of Treg could lead to serious infections or the appearance of cancer. However, in human bone marrow transplantation adoptive transfer of Treg has already been used to prevent graftversus-host disease and in these clinical trials Treg transfer seems to be safe [45]. In addition, Treg homing to the inflamed liver has to be guaranteed. Another point is Treg plasticity since it has been shown that Treg are able to convert into proinflammatory cell types and could possibly aggravate inflammation [33]. In vitro it was possible to prevent the conversion of in-vitro-induced Treg into proinflammatory Th17 cells by blocking IL-17-relevant cytokines [46]. Nonetheless, it remains unclear whether transferred Treg will keep their suppressive character once they reach a solid organ with ongoing autoimmune inflammation.
Discussion
As in other autoimmune diseases, a dysbalance between Treg and proinflammatory T cells has been proposed for the pathogenesis of AIH. Selective immunomodulatory therapy is an attractive option to avoid general immunosuppression and other side effects. If Treg impairment is pathogenic for AIH, adaptive transfer of this cell population could be a reasonable therapeutic apSebode/Lohse
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clear that only cells with the highest expression of CD25 (CD25high) possess suppressive capacity [29]. Thereafter, the intracellular transcription factor Foxp3 has been regarded as the most reliable Treg marker. Unfortunately, in humans Foxp3 can be expressed transiently by T-effector cells [30]. Thus, a combination of several markers seems to be the best approach to define Treg. Indeed, by using such a combination, we have recently been able to show that peripheral Treg frequency is not impaired in adult AIH patients [31]. Instead, peripheral and intrahepatic Treg frequency correlated with disease activity. This is further supported by a report which showed that Treg frequency is generally elevated in inflamed livers irrespective of its cause [32]. If there is not a global defect of Treg numbers in AIH, the question rises why the attracted Treg are not able to suppress hepatic inflammation? Maybe the inflamed microenvironment of the liver hampers their suppressive function. Inflammation could possibly also impact on Treg plasticity with the consequence of Treg adopting a more proinflammatory rather than a tolerant character [33].
proach. It is still unclear whether Treg are globally impaired and play a major role for the pathogenesis of AIH. Besides, the trigger that breaks hepatic tolerance as well as the main autoantigen for AIH are still elusive. If Treg transfer only corrects an epiphenomenon, it is unlikely to become a promising therapy. In addition, it would carry the risk of disease aggravation if conversion of Treg into proinflammatory cells cannot be avoided. This leaves us
with the final and crucial question of whether we understand the pathogenesis of AIH to such an extent that we are ready for selective immunotherapy.
Disclosure Statement The authors do not have any conflicts of interest to disclose.
References
Future Perspective: Immunomodulatory Therapy for Autoimmune Hepatitis
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29 Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA: CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001; 167:1245–1253. 30 Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF: Induction of FoxP3 and acquisition of T-regulatory activity by stimulated human CD4+CD25– T cells. J Clin Invest 2003; 112:1437–1443. 31 Peiseler M, Sebode M, Franke B, Wortmann F, Schwinge D, Quaas A, Baron U, Olek S, Wiegard C, Lohse AW, Weiler-Normann C, Schramm C, Herkel J: FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency. J Hepatol 2012;57:125–132. 32 Speletas M, Argentou N, Germanidis G, Vasiliadis T, Mantzoukis K, Patsiaoura K, Nikolaidis P, Karanikas V, Ritis K, Germenis AE: Foxp3 expression in liver correlates with the degree but not the cause of inflammation. Mediators Inflamm 2011;2011:827565. 33 Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK: Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235–238. 34 Luther C, Adamopoulou E, Stoeckle C, Brucklacher-Waldert V, Rosenkranz D, Stoltze L, Lauer S, Poeschel S, Melms A, Tolosa E: Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients. J Immunol 2009;183:841–848.
Future Perspective: Immunomodulatory Therapy for Autoimmune Hepatitis Marcial Sebode Ansgar W. Lohse I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Abstract In the last two decades, more and more light has been shed on the immunologic pathogenesis of autoimmune liver diseases, notably autoimmune hepatitis (AIH). An immunologic dysbalance with proinflammatory immune responses dominating over hepatic tolerance seems to be part of AIH pathogenesis. In detail, an impairment of regulatory T cells (Treg) is suspected. If this holds true and reduced Treg numbers or their function are pathogenic for AIH, this offers the option of cellular or immunomodulatory therapy. However, the exact immunological role of Treg in AIH still needs to be clarified before cellular therapy is promising for patients. © 2014 S. Karger AG, Basel
Introduction
The liver is continuously exposed to antigens provided by portal venous blood flow. To avoid overwhelming inflammatory responses, a network of hepatic immune cells preserves the balance between tolerance and defense against infectious diseases (fig. 1). The hepatic immune microenvironment turns the liver into a tolerant organ © 2014 S. Karger AG, Basel 0257–2753/14/0325–0502$39.50/0 E-Mail [email protected] www.karger.com/ddi
[1]. This is evident, for example, from the well-known observation that transplanted livers are better tolerated than other organs [2]. Hepatic tolerance also impacts on other inflamed organs: in the mouse model of multiple sclerosis the clinical phenotype was ameliorated once the neuronal autoantigen was expressed in the liver [3]. In the state of autoimmune liver disease a dysbalance has been suggested which may account for an autoimmune reaction against hepatocytes in autoimmune hepatitis (AIH) or against the biliary tree in primary biliary cirrhosis and in primary sclerosing cholangitis. Research has mainly concentrated on the impaired immune regulation in AIH. Therefore, this article will focus on the disrupted immune homeostasis in AIH and discuss the option of immunomodulatory treatment.
Immunological Dysbalance in AIH
AIH is a chronic inflammatory disease of the liver that is characterized by elevated transaminases and raised immunoglobulins, notably of immunoglobulin G, as well as autoantibodies [reviewed in 4, 5]. The autoimmune etiology of AIH is widely accepted. The disease occurs predominantly in women and in most cases remission can be induced by immunosuppressive treatment. However, the reason why hepatic tolerance is broken and which immune reactions are specific for AIH is not yet fully understood. Prof. Dr. A.W. Lohse I. Department of Medicine University Medical Center Hamburg-Eppendorf Martinistrasse 52, DE–20246 Hamburg (Germany) E-Mail alohse @ uke.de
Downloaded by: University of Iowa Libraries 128.255.6.125 - 5/31/2015 7:15:17 PM
Key Words Autoimmune hepatitis · Regulatory T cells · Immune regulation · Cellular therapy
Fig. 1. Immune homeostasis in AIH. Self-antigens are presented to the immune system by hepatocytes or other antigen-presenting cells (APC). In AIH, effector T cells including CD4+, CD8+ or Th17 cells recognize these antigens and promote liver inflammation. Treg that are probably antigen-specific normally limit hepatic inflammation.
Effector cells mediate the inflammation of autoimmune liver disease. First CD4+ and CD8+ T cells were recognized to be responsible for hepatocellular damage. A CD4+ or Th1 immune response was favored in two different mouse models of AIH [14, 15]. In another model and in peripheral blood of AIH patients, CD8+ T cells were the main effector cell type [16, 17]. Th17 cells, a Thelper cell subtype that is characterized by proinflammatory interleukin (IL)-17, appear to be involved in the pathogenesis of autoimmune diseases, e.g. multiple sclerosis or rheumatoid arthritis [18, 19]. Even in autoimmune liver diseases there seems to be a role for IL-17-producing cells [20–22]. If IL-17 plays a major role in autoimmune liver disease, a neutralizing IL-17 antibody could offer specific blockade of liver inflammation [23] and avoid general immunosuppression by standard therapy. However, an unsolved question is whether hepatic IL17-producing cells are pathogenic for autoimmune liver diseases or whether they solely represent an inflammatory bystander reaction [24]. Besides CD8+, Th1 and Th17 cells, innate immune cells such as NK cells or unconventional T cells like γδ T and NKT cells could play a role in disrupted immune homeostasis of AIH, but data on these cell types is limited.
It is assumed that tolerance to liver antigens is lost in AIH, which leads to the activation of autoreactive T cells. Specific T cells have been identified to react with CYP2D6 or SepSecS autoantigens [6, 7]. Still, except for the abovementioned autoantigens in type 2 or anti-SLA/LP (soluble liver antigen/liver pancreas antigen)-positive type 1 AIH, the main autoantigens of AIH remain elusive. Antigen presentation is mediated by human leukocyte antigens (HLA). An immunogenic predisposition is reflected by the association of AIH with HLA subtypes, e.g. type 1 AIH is preferentially associated with HLA-DRB1*0301 and HLA-DRB1*0401 [8]. HLA subtypes have an influence on the therapeutic response and clinical course [9]. As an important part of the hepatic microenvironment, antigen-presenting cells can shape immune reactions [1, 10]. Hepatic antigen-presenting cells comprise hepatocytes, Kupffer cells, dendritic cells, stellate cells and liver sinusoidal endothelial cells. Antigen-presenting cells like liver sinusoidal endothelial cells could play a role in AIH by modulating T-effector cell responses or induction of tolerance [11–13].
Apart from identifying the pathogenic proinflammatory responses, the impaired anti-inflammatory immunoregulation in AIH has been thoroughly investigated. Regulatory T cells (Treg) are important mediators of immune tolerance and their impairment seems to be involved in autoimmune diseases [25]. Treg are capable of suppressing the effector functions of various cell types. Hints for a role of Treg in AIH come from a mouse model of spontaneous liver inflammation which combined the loss of Treg with the disruption of immunoregulatory signalling by the molecule ‘PD-1’ (programmed cell death-1) [26]. Several studies from King’s College London propose a global impairment of Treg function and numbers in pediatric AIH patients [reviewed in 27]. In a very recent publication, Grant et al. [28] showed that Treg from AIH patients fail to suppress proinflammatory IL17 by CD4+ T cells. Still, the role of Treg in the pathogenesis of AIH is not entirely clear. One reason lies in the fact that specific definition of Treg by intra- and extracellular markers is challenging. In early studies, Treg were defined as CD4+CD25+ T cells. In the following years it became
Future Perspective: Immunomodulatory Therapy for Autoimmune Hepatitis
Dig Dis 2014;32:502–506 DOI: 10.1159/000360493
503
Downloaded by: University of Iowa Libraries 128.255.6.125 - 5/31/2015 7:15:17 PM
Regulatory T Cells in AIH
Immunomodulatory Therapy for AIH
Standard immunosuppressive treatment for AIH consists of corticosteroids in combination with azathioprine. Thereby, remission is induced in most patients. This suggests that T-effector cells are limited by immunosuppression. Whether azathioprine and prednisolone play an auxiliary immunomodulatory role in AIH is unclear. This has been proposed for prednisolone in other autoimmune diseases like myasthenia gravis [34]. However, the pathogenic immune reaction of AIH is not terminated by classical immunosuppressive therapy. That is why recurrent courses of AIH can occur after tapering immunosuppression. A more selective therapy for AIH is highly desirable. Immunomodulatory approaches for AIH have mainly focused on the Treg population. In order to support tolerance in the liver, efforts to stabilize or even enlarge Treg have been undertaken. The mTOR inhibitor rapamycin or the vitamin A metabolite retinoic acid can promote Treg numbers [35, 36]. Another agent supporting Treg recovery is IL-2 [37]. In pediatric patients with primary sclerosing cholangitis and inflammatory bowel disease, the oral antibiotic vancomycin had immunomodulatory effects and resulted in increased peripheral Treg numbers and normalization of liver function tests [38]. Secondline therapy for AIH such as anti-TNF-α treatment could 504
Dig Dis 2014;32:502–506 DOI: 10.1159/000360493
also imply immunomodulation [39]. It has been shown that anti-TNF-α treatment reversed the reduction of peripheral Treg numbers and their increased apoptosis in inflammatory bowel disease [40]. To create large numbers of Treg, the best approach seems to be the adoptive transfer of ex vivo expanded Treg. In a mouse model of type 2 AIH, transferred antigen-specific Treg were able to restore peripheral tolerance [41]. This was confirmed in humans by Longhi et al. [42] who generated CYP2D6-specific Treg that efficiently suppressed in vitro autoreactive CD4+ T cells derived from type 2 AIH patients. In general, mouse models have shown that antigen-specific Treg are more effective in suppressing autoimmune diseases than polyclonal Treg [reviewed in 43]. Unfortunately, in most cases of type 1 AIH the autoantigen is unknown and therefore only polyclonal Treg are available for adoptive transfer. It is uncertain whether in human autoimmune diseases polyclonal Treg are capable of suppressing effector cells. Several technical problems still need to be overcome before adoptive transfer of Treg becomes applicable for clinical studies in AIH [reviewed in 44]. One general concern is safety since the administration of Treg could lead to serious infections or the appearance of cancer. However, in human bone marrow transplantation adoptive transfer of Treg has already been used to prevent graftversus-host disease and in these clinical trials Treg transfer seems to be safe [45]. In addition, Treg homing to the inflamed liver has to be guaranteed. Another point is Treg plasticity since it has been shown that Treg are able to convert into proinflammatory cell types and could possibly aggravate inflammation [33]. In vitro it was possible to prevent the conversion of in-vitro-induced Treg into proinflammatory Th17 cells by blocking IL-17-relevant cytokines [46]. Nonetheless, it remains unclear whether transferred Treg will keep their suppressive character once they reach a solid organ with ongoing autoimmune inflammation.
Discussion
As in other autoimmune diseases, a dysbalance between Treg and proinflammatory T cells has been proposed for the pathogenesis of AIH. Selective immunomodulatory therapy is an attractive option to avoid general immunosuppression and other side effects. If Treg impairment is pathogenic for AIH, adaptive transfer of this cell population could be a reasonable therapeutic apSebode/Lohse
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clear that only cells with the highest expression of CD25 (CD25high) possess suppressive capacity [29]. Thereafter, the intracellular transcription factor Foxp3 has been regarded as the most reliable Treg marker. Unfortunately, in humans Foxp3 can be expressed transiently by T-effector cells [30]. Thus, a combination of several markers seems to be the best approach to define Treg. Indeed, by using such a combination, we have recently been able to show that peripheral Treg frequency is not impaired in adult AIH patients [31]. Instead, peripheral and intrahepatic Treg frequency correlated with disease activity. This is further supported by a report which showed that Treg frequency is generally elevated in inflamed livers irrespective of its cause [32]. If there is not a global defect of Treg numbers in AIH, the question rises why the attracted Treg are not able to suppress hepatic inflammation? Maybe the inflamed microenvironment of the liver hampers their suppressive function. Inflammation could possibly also impact on Treg plasticity with the consequence of Treg adopting a more proinflammatory rather than a tolerant character [33].
proach. It is still unclear whether Treg are globally impaired and play a major role for the pathogenesis of AIH. Besides, the trigger that breaks hepatic tolerance as well as the main autoantigen for AIH are still elusive. If Treg transfer only corrects an epiphenomenon, it is unlikely to become a promising therapy. In addition, it would carry the risk of disease aggravation if conversion of Treg into proinflammatory cells cannot be avoided. This leaves us
with the final and crucial question of whether we understand the pathogenesis of AIH to such an extent that we are ready for selective immunotherapy.
Disclosure Statement The authors do not have any conflicts of interest to disclose.
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Future Perspective: Immunomodulatory Therapy for Autoimmune Hepatitis
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