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Hepatology 75(1), 13-27 (2022). doi:10.1002/hep.32134, Published by Wolters Kluwer Health, [Alphen aan den Rijn]

The constant exposure of the liver to gut derived foreign antigens has resulted in this organ attaining unique immunological characteristics, however it remains susceptible to immune mediated injury. Our understanding of this type of injury, in both the native and transplanted liver, has improved significantly in recent decades. This includes a greater awareness of the tolerance inducing CD4+CD25+CD127lowT-cell lineage with the transcription factor FoxP3, known as regulatory T-Cells (Tregs). These cells comprise 5-10% of CD4+T cells and are known to function as an immunological “braking” mechanism, thereby preventing immune mediated tissue damage. Therapies that aim to increase Treg frequency and function have proved beneficial in the setting of both autoimmune diseases and solid organ transplantations. The safety and efficacy of Treg therapy in liver disease is an area of intense research at present and has huge potential. Due to these cells possessing significant plasticity, and the potential for conversion towards a T-helper 1 (Th1) and 17 (Th17) subsets in the hepatic microenvironment, it is pre-requisite to modify the microenvironment to a Treg favourable atmosphere to maintain these cells’ function. In addition, implementation of therapies that effectively increase Treg functional activity in the liver may result in the suppression of immune responses and will hinder those that destroy tumour cells. Thus, fine adjustment is crucial to achieve this immunological balance. This review will describe the hepatic microenvironment with relevance to Treg function, and the role these cells have in both native diseased and transplanted livers.

The tight relationship between the gut and liver on embryological, anatomical and physiological levels inspired the concept of a gut-liver axis as a central element in the pathogenesis of gut-liver axis diseases. This axis refers to the reciprocal regulation between these two organs causing an integrated system of immune homeostasis or tolerance breakdown guided by the microbiota, the diet, genetic background, and environmental factors. Continuous exposure of gut microbiome, various hormones, drugs and toxins, or metabolites from the diet through the portal vein adapt the liver to maintain its tolerogenic state. This is orchestrated by the combined effort of immune cells network: behaving as a sinusoidal and biliary firewall, along with a regulatory network of immune cells including, regulatory T cells and tolerogenic dendritic cells (DC). In addition, downregulation of costimulatory molecules on hepatic sinusoids, hepatocytes and biliary epithelial cells as well as regulating the bile acids chain also play a part in hepatic immune homeostasis. Recent evidence also demonstrated the link between changes in the gut microbiome and liver resident immune cells in the progression of cirrhosis and the tight correlation among primary sclerosing cholangitis (PSC) and also checkpoint induced liver and gut injury. In this review, we will summarize the most recent evidence of the bidirectional relationship among the gut and the liver and how it contributes to liver disease, focusing mainly on PSC and checkpoint induced hepatitis and colitis. We will also focus on completed therapeutic options and on potential targets for future treatment linking with immunology and describe the future direction of this research, taking advantage of modern technologies.

Background & Aims Autoimmune hepatitis (AIH) is an immune-mediated disease with no curative treatment. Regulatory T cell (Treg) therapy is potentially curative in AIH given the critical role of Tregs in preventing autoimmunity. To work effectively, adoptively transferred Tregs must migrate to and survive within the inflamed liver. We conducted a proof-of-concept study aiming to assess the safety and liver-homing properties of good manufacturing practice (GMP)-grade autologous Tregs in patients with AIH. Methods Autologous polyclonal GMP-grade Tregs were isolated using leukapheresis and CliniMACS, labelled with indium tropolonate and re-infused intravenously to 4 patients with AIH. GMP-Treg homing to the liver was investigated with longitudinal gamma camera and SPECT-CT scanning. GMP-Treg immunophenotype, function and immunometabolic state were assessed during the study. Results We observed that the isolated Tregs were suppressive and expressed CXCR3, a chemokine receptor involved in recruitment into the inflamed liver, as well as Treg functional markers CD39, CTLA-4 and the transcription factor Foxp3. Serial gamma camera and SPECT-CT imaging demonstrated that 22–44% of infused Tregs homed to and were retained in the livers of patients with autoimmune hepatitis for up to 72 h. The infused cells did not localise to any off-target organs other than the spleen and bone marrow. GMP-Tregs were metabolically competent and there were no infusion reactions or high-grade adverse effects after Treg infusion. Conclusion Our novel findings suggest that the liver is a good target organ for Treg cellular therapy, supporting the development of clinical trials to test efficacy in autoimmune hepatitis and other autoimmune liver diseases. Lay summary Autoimmune liver diseases occur when the body’s immune cells target their own liver cells. Regulatory T cells (Tregs) prevent autoimmunity, thus they are a potential therapy for autoimmune liver diseases. In patients with autoimmune hepatitis, Treg infusion is safe, with nearly a quarter of infused Tregs homing to the liver and suppressing tissue-damaging effector T cells. Thus, Tregs are a potentially curative immune cell therapy for early autoimmune liver diseases., Graphical abstract Unlabelled Image, Highlights • Tregs from patients with autoimmune hepatitis are suppressive, possess functional markers CD39 and CTLA-4, and express CXCR3. • Treg infusion in autoimmune liver disease is safe without any side effects. • 22-44% of infused Tregs home to and were retained in the livers of patients with autoimmune hepatitis for up to 72 hours. • GMP-Tregs from patients with autoimmune hepatitis were metabolically competent.

Human liver possesses a persistent and tightly regulated immune response. Maintaining this homeostatic state is the key to prevent pathological processes, as a failure in clearing dangerous stimuli, is associated with tissue damage. A dysregulation of the liver immune homeostasis is involved in many disease processes and the use of the immunosuppression aims to control the inflammatory response, where the physiologic mechanisms failed. The use of steroids which targets broadly the inflammatory cascade and the immune system activation have been extensively employed in both acute and chronic liver diseases. They currently are the backbone of the treatment of autoimmune diseases such as autoimmune hepatitis or IgG4 sclerosing cholangitis. The steroid use in acute liver injury, especially alcohol mediated and drug induced liver injury (DILI), have been debated, despite the biological rationale. The immunosuppression molecules currently employed in liver diseases target the immune system broadly, causing multiple side effects either intrinsic in the mechanisms of the drug or secondary to off-target toxicity. The future of immunosuppressant treatment is moving towards more selective strategies, targeting disease specific pathways. This review aims to explore the rationale of use of immunosuppression in non-transplant hepatology. A broad summary of the immune biology of liver immune mediated diseases will be provided to the readers in order to highlight the potential therapeutic targets. An extensive description of the molecules employed in liver diseases will follow and the clinical evidences in AIH, IgG4 related cholangitis, alcoholic hepatitis and DILI will be reviewed.

The liver is a complex immunological organ. It has both immunogenic and tolerogenic capacity. Tolerogenic potential of human liver with its protective firewalls is required to guard the body against the continuous influx of microbial product from the gut via the sinusoids and biliary tree. Immunotolerance and anergic state is maintained by a combined effort of both immune cells, parenchyma cells, epithelial and endothelial cells. Despite this, an unknown trigger can ignite the pathway towards breakdown in hepatic tolerance leading to autoimmune liver diseases. Understanding the initial stimulus which causes the hepatic immune system to switch from the regulatory arm towards self-reactive effector arm remains challenging. Dissecting this pathology using the current technological advances is crucial to develop curative immune based therapy in autoimmune liver diseases. We discuss the hepatic immune cells and non-immune cells which maintain liver tolerance and the evidence of immune system barrier breach which leads to autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis.

Regulatory T cells (Tregs) are crucial in maintaining tolerance. Hence, Treg immunotherapy is an attractive therapeutic option in autoimmune diseases and organ transplantations. Currently, autoimmune diseases do not have a curative treatment and transplant recipients require life-long immunosuppression to prevent graft rejection. There has been significant progress in understanding polyclonal and antigen-specific Treg biology over the last decade. Clinical trials with good manufacturing practice (GMP) Treg cells have demonstrated safety and early efficacy of Treg therapy. GMP Treg cells can also be tracked following infusion. In order to improve efficacy of Tregs immunotherapy, it is necessary that Tregs migrate, survive and function at the specific target tissue. Application of antigen specific Tregs and maintaining cells' suppressive function and survival with low dose interleukin-2 (IL-2) will enhance the efficacy and longevity of infused GMP-grade Tregs. Notably, stability of Tregs in the local tissue can be manipulated by understanding the microenvironment. With the recent advances in GMP-grade Tregs isolation and antigen-specific chimeric antigen receptor (CAR)-Tregs development will allow functionally superior cells to migrate to the target organ. Thus, Tregs immunotherapy may be a promising option for patients with autoimmune diseases and organ transplantations in near future.

The induction of graft tolerance remains the holy grail of transplantation. This is important as chronic allograft dysfunction and the side effects of immunosuppression regimens place a major burden on the lives of transplant patients and their healthcare systems. This has mandated the need to understand the immunobiology of graft rejection and identify novel therapeutics. Regulatory T (Treg) cells play an important role in modulating pro‐inflammatory microenvironments and maintaining tissue homeostasis. However, there are fundamental unanswered questions regarding Treg cell immunobiology. These cells are a heterogeneous entity with functionally diverse roles. Moreover, the adoption of novel deeper immunophenotyping and genomic sequencing technologies has identified this phenotype and function to be more complex than expected. Hence, a comprehensive understanding of Treg cell heterogeneity is needed to safely and effectively exploit their therapeutic potential. From a clinical perspective, the recent decade has seen different clinical teams commence and complete first‐in‐man clinical trials utilising Treg cells as an adoptive cellular therapy. In this review, we discuss these trials from a translational perspective with an important focus on safety. Finally, we identify crucial knowledge gaps for future study., Treg cell therapy has the potential to revolutionise current post‐transplant immunosuppression protocols. Treg cells are presently undergoing early phase trials in solid organ transplantation. However, there are multiple questions regarding Treg cell immunobiology that need to be resolved.

Primary biliary cholangitis (PBC) is a cholestatic liver disease characterized by the destruction of the small and medium bile ducts. Its pathogenesis is still unknown. Despite the genome wide association study findings, the therapies targeting the cytokines pathway, tested so far, have failed. The concept of the biliary epithelium as a key player of the PBC pathogenesis has emerged over the last few years. It is now well accepted that the biliary epithelial cells (BECs) actively participate to the genesis of the damage. The chronic stimulation of BECs via microbes and bile changes the cell phenotype toward an active state, which, across the production of proinflammatory mediators, can recruit, retain, and activate immune cells. The consequent immune system activation can in turn damage BECs. Thus, the crosstalk between both innate and adaptive immune cells and the biliary epithelium creates a paracrine loop responsible for the disease progression. In this review, we summarize the evidence provided in literature about the role of BECs and the immune system in the pathogenesis of PBC. We also dissect the relationship between the immune system and the BECs, focusing on the unanswered questions and the future potential directions of the translational research and the cellular therapy in this area.

The gut–liver axis is increasingly considered to play a vital part in the progression of chronic inflammatory gut and liver diseases. Hence, a detailed understanding of the local and systemic regulatory mechanisms is crucial to develop novel therapeutic approaches. In this review, we discuss in-depth the roles of regulatory T cells (Tregs) and mucosal-associated invariant T cells (MAITs) within the context of inflammatory bowel disease, primary sclerosing cholangitis, and non-alcoholic steatohepatitis. Tregs are crucial in maintaining peripheral tolerance and preventing autoimmunity. MAIT cells have a unique ability to rapidly recognize microbial metabolites and mount a local immune response and act as a ‘biliary firewall’ at the gut and biliary epithelial barrier. We also outline how current knowledge can be exploited to develop novel therapies to control the propagation of chronic gut- and liver-related inflammatory and autoimmune conditions. We specifically focus on the nature of the Tregs’ cell therapy product and outline an adjunctive role for low-dose IL-2. All in all, it is clear that translational immunology is at crucial crossroads. The success of ongoing clinical trials in cellular therapies for inflammatory gut and liver conditions could revolutionize the treatment of these conditions and the lives of our patients in the coming years. Electronic supplementary material The online version of this article (10.1007/s12072-018-9882-x) contains supplementary material, which is available to authorized users.

Background/aimsIntestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown.Methods/designWe analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively.ResultsIn ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments.ConclusionsIn ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the ‘leaky’ gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

Background Autoimmune hepatitis (AIH) is an immune-mediated liver disease of unknown etiology. Increasing incidence of AIH in Asian patients has been reported. However, the phenotypic difference of Asian patients in Europe and Asia has still not been explored. Aim To evaluate the clinical presentation, biochemical and immunological profiles, treatment response and survival outcome of type 1 AIH from two tertiary liver transplant centres (United Kingdom and Singapore). Method Patients who fulfilled the simplified diagnostic scoring criteria of AIH were included in the study. Patients with overlap syndrome were excluded. Results Totals of 40 Asian patients and 159 Caucasian patients from the University Hospital of Birmingham National Health Service Foundation Trust, UK, were compared with 57 Asian patients from Singapore General Hospital, Singapore. Asian patients from Singapore present significantly much later (median 55 vs. 32 years, p

The human liver is regarded as a lymphoid organ that contributes to both local and systemic immune response. Intrahepatic immune cells including regulatory T cells (Tregs) reside in the hepatic microenvironment which is enriched with proinflammatory cytokines, chemokines and metabolites. In addition, the hepatic microenvironment has the unique ability to establish and maintain immune tolerance despite the continuous influx of the gut derived microbial products via the portal vein. Regulatory T cells play a crucial role in maintaining the hepatic tolerogenic state; however, the phenotypic stability, function and survival of Tregs in the inflamed liver microenvironment is still poorly understood. Despite this, Tregs immunotherapy remains as an appealing therapeutic option in autoimmune and immune mediated liver diseases. In order to advance cell therapy, it is important for us to further our understanding of the hepatic microenvironment, with the aim of developing ways to modify the hostile, inflamed environment to one which is more favourable. By doing so, T cell stability and function would be enhanced, resulting in improved clinical outcomes.

Autoimmune hepatitis (AIH) is an immune-mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom-AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3-positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C-X-C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme-linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin-like transcript-1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon-γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity-associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin-like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment-naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421-436).

There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6+CD4+ and AhR+CD4+ T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1β. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies.

Thymic-derived naturally occurring regulatory T cells (tTreg) are crucial for maintaining peripheral immune homeostasis. They play a crucial role in preventing autoimmunity and maintaining organ transplant without requiring immunosuppression. Cellular metabolism has recently emerged as an important regulator of adaptive immune cell balance between Treg and effector T cells. While the metabolic requirements of conventional T cells are increasingly understood, the role of Treg cellular metabolism is less clear. The continuous exposure of metabolites and nutrients to the human liver via the portal blood flow influences the lineage fitness, function, proliferation, migration and survival of Treg cells. As cellular metabolism has an impact on its function, it is crucial to understand the metabolic pathways wiring in regulatory T cells. Currently, there are ongoing early phase clinical trials with polyclonal and antigen-specific good manufacturing practice (GMP) Treg therapy to treat autoimmune diseases and organ transplantation. Thus, enhancing immuno-metabolic pathways of Treg cells by translational approach with existing or new drugs would utilize Treg cells to their full potential for effective cellular therapy.

Hepatitis B virus (HBV) infection is a global health problem. About 400 million people are chronically infected with HBV, which is a major cause of liver cirrhosis and liver failure. The global distribution has changed due to migration. Immunization programs have resulted in a dramatic decline in the incidence and prevalence of HBV and hepatitis D virus (HDV) infection in many countries. Host immune responses to the viruses define the clinical course of infection, which varies from an asymptomatic carrier state to fulminant hepatic failure. The timing of treatment and the choice of antiviral therapy depend on the patient profile. The main goal of antiviral therapy is to prevent liver cirrhosis. Antiviral treatment can suppress HBV to undetectable levels. However, the HBV genome can persist in hepatocytes despite sustained and potent suppression. HDV requires co-existing HBV for its proliferation. Around 20 million people are co-infected with HDV and HBV. In general, HBV/HDV co-infection causes more severe liver disease than is observed in HBV alone. Interferon is the current accepted treatment option of HBV/HDV infection, though treatment results are disappointing. For selected patients with decompensated liver cirrhosis due to HBV or HBV/HDV infection, liver transplantation may be appropriate.

Introduction Autoimmune hepatitis is a chronic immune-mediated liver injury, frequently associated with progression to end-stage liver disease if untreated. Patients commonly present with hepatitis, positive immune serology, elevated immunoglobulins and compatible liver histology, in the absence of an alternative aetiology. Sources of data Data for this review were obtained using PubMed. Areas of agreement Disease usually responds to steroids and azathioprine, and appears to be a manifestation of autoimmune predisposition triggered in genetically susceptible individuals exposed to likely environmental challenges. We provide an up-to-date approach to disease understanding and management along with the clinical approach to diagnosis and current treatment suggestions. Areas of controversy Controversies such as second line therapies and novel markers of disease activity are introduced. Growing points Increased understanding of the immunoregulatory mechanisms behind autoimmune hepatitis has led to opportunities for new therapies. These are developed including a discussion of timely research studies relevant to future therapies for patients.

IntroductionInnate lymphoid cells (ILC) have been implicated in the initiation of inflammation and fibrosis in mice. However, ILC have not been characterized in inflamed human liver tissue.MethodsHuman intrahepatic lymphocytes were isolated by mechanical digestion and phenotyped by flow cytometry. Conditioned medium from cultures of primary human biliary epithelial cells, stellate cells, fibroblasts and inflamed human liver tissue was used to model the effects of the inflammatory liver environment of ILC phenotype and function.ResultsAll three ILC subsets were present in the human liver, with the ILC1 (CRTH2negCD117neg) subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44+) and NCRneg ILC3 (CRTH2negCD117pos) subsets were also detected. ILC2 (CRTH2pos) frequency correlated with disease severity measured by model of end stage liver disease (MELD) scoring leading us to study this subset in more detail. ILC2 displayed a tissue resident CD69+ CD161++ phenotype and expressed chemokine receptor CCR6 allowing them to respond to CCL20 secreted by cholangiocytes and stellate cells. ILC2 expressed integrins VLA-5 and VLA-6 and the IL-2 and IL-7 cytokine receptors CD25 and CD127 although IL-2 and IL-7 were barely detectable in inflamed liver tissue. Although biliary epithelial cells secrete IL-33, intrahepatic ILC2 had low expression of the ST2 receptor. Intrahepatic ILC2 secreted the immunoregulatory and repair cytokines IL-13 and amphiregulin.ConclusionsIntrahepatic ILC2 express receptors allowing them to be recruited to bile ducts in inflamed portal tracts. Their frequencies increased with worsening liver function. Their secretion of IL-13 and amphiregulin suggests they may be recruited to promote resolution and repair and thereby they may contribute to ongoing fibrogenesis in liver disease.

Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3(+) regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by "omics" and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients.

The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg). The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis, or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients. Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment, and good manufacturing practice facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases (AILD), chronic rejection, and posttransplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases, and solid organ transplantations. There have not been any new therapies for the AILD for more than three decades; thus, the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior, and microenvironment of Treg before applying the cells to the patients.

Hepatitis C virus infection is a major cause of chronic liver disease. CD4(+) T cells play a key role in disease outcome. However, the critical functions and associated phenotypes of intrahepatic CD4(+) T cells are not well defined. We have previously shown that CD8(+) T cells expressing the C type lectin CD161 are highly enriched in the human liver, especially during chronic hepatitis. These cells are associated with a type 17 differentiation pattern and express cytokines including IL-17A, IL-22, and IFN-γ. We therefore analyzed expression of CD161 on CD4(+) T cells in blood and liver and addressed the relevant phenotype and functional capacity of these populations. We observed marked enrichment of CD161(+)CD4(+) T cells in the liver during chronic hepatitis such that they are the dominant subtype (mean 55% of CD4(+) T cells). IL-22 and IL-17 secreting CD4(+) T cells were readily found in the livers of HCV(+) and NASH donors, although not enriched compared to blood. There was, however, specific enrichment of a novel subset of IL-22/IFN-γ dual secretors (p = 0.02) compared to blood, a result reconfirmed with direct ex vivo analyses. These data indicate the dominance of CD161(+) expressing lymphocyte populations within the hepatic infiltrate, associated with a distinct cytokine profile. Given their documented roles as antiviral and hepatoprotective cytokines respectively, the impact of co-secretion of IFN-γ and IL-22 in the liver may be particularly significant.

The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg). The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis, or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients. Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment, and good manufacturing practice facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases (AILD), chronic rejection, and posttransplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases, and solid organ transplantations. There have not been any new therapies for the AILD for more than three decades; thus, the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior, and microenvironment of Treg before applying the cells to the patients.

Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3+regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by “omics” and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients.

Kupffer cells are liver macrophages abundantly resident in the sinusoids and are the largest tissue-resident macrophage population. They may arise from resident progenitor cells or infiltrating myeloid cells. They are important for homeostasis in the liver and for protective immune responses, a combination of traits that makes them an ideal vascular firewall in the liver. Their tolerogenic phenotype may, however, be exploited in disease processes such as malaria infection. Their functions include phagocytosis of foreign antigens, antigen presentation to T cells, and secretion of several key inflammatory mediators which further extend the immune repertoire of Kupffer cells. We discuss the established roles of Kupffer cells in immunity.

Liver fibrosis is a wound healing response to chronic liver injury and inflammation in which macrophages and infiltrating monocytes participate in both the development and resolution phase. In humans, three monocyte subsets have been identified: the classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. We studied the phenotype and function of these monocyte subsets in peripheral blood and liver tissue from patients with chronic inflammatory and fibrotic liver diseases. The frequency of intrahepatic monocytes increased in disease compared with control liver tissue, and in both nondiseased and diseased livers there was a higher frequency of CD14++CD16+ cells with blood. Our data suggest two nonexclusive mechanisms of CD14++CD16+ accumulation in the inflamed liver: (1) recruitment from blood, because more than twice as many CD14++CD16+ monocytes underwent transendothelial migration through hepatic endothelial cells compared with CD14++CD16− cells; and (2) local differentiation from CD14++CD16− classical monocytes in response to transforming growth factor β and interleukin (IL)-10. Intrahepatic CD14++CD16+ cells expressed both macrophage and dendritic cell markers but showed high levels of phagocytic activity, antigen presentation, and T cell proliferation and secreted proinflammatory (tumor necrosis factor α, IL-6, IL-8, IL-1β) and profibrogenic cytokines (IL-13), chemokines (CCL1, CCL2, CCL3, CCL5), and growth factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor), consistent with a role in the wound healing response. Conclusion: Intermediate CD14++CD16+ monocytes preferentially accumulate in chronically inflamed human liver as a consequence of enhanced recruitment from blood and local differentiation from classical CD14++CD16− monocytes. Their phagocytic potential and ability to secrete inflammatory and profibrogenic cytokines suggests they play an important role in hepatic fibrogenesis. (HEPATOLOGY 2013)

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3+ regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1– and VAP-1–rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.

Autoimmune hepatitis (AIH), primary biliary cirrhosis, and primary sclerosing cholangitis are the three major autoimmune diseases affecting the liver, and of these three, AIH is the most typical autoimmune disease being characterized by a T-cell-rich infiltrate, raised circulating γ-globulins, autoantibodies, HLA associations, and links with other autoimmune diseases. It is the only one, of the three diseases, that responds well to immunosuppressive therapy. AIH is caused by dysregulation of immunoregulatory networks and the consequent emergence of autoreactive T cells that orchestrate a progressive destruction of hepatocytes leading untreated to liver failure. T cells play a major role in the immunopathogenesis, and both CD4(+) and CD8(+) T cells are involved together with effector responses mediated by NK cells, γδ T cells, and macrophages. A number of triggering factors have been proposed including viruses, xenobiotics, and drugs, but none have been conclusively shown to be involved in pathogenesis.

Regulatory T cells (Tregs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of Treg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)+ Tregs. We isolated CD4+CD25+CD127lowFoxP3+ Tregs from chronically inflamed human liver removed at transplantation; compared with blood-derived Tregs, liver-derived Tregs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, Tregs used CXCR3 and α4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4+ Tregs in the parenchyma and septal areas. Ex vivo, liver-derived Tregs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of Tregs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit Tregs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of Tregs at sites of hepatitis in chronic liver disease.

Liver inflammation is greater in nonalcoholic steatohepatitis (NASH) than steatosis, suggesting that immune responses contribute to nonalcoholic fatty liver disease (NAFLD) progression. Livers normally contain many natural killer T (NKT) cells that produce factors that modulate inflammatory and fibrogenic responses. Such cells are relatively depleted in steatosis, but their status in more advanced NAFLD is uncertain. We hypothesized that NKT cells accumulate and promote fibrosis progression in NASH. We aimed to determine if livers become enriched with NKT cells during NASH-related fibrosis; identify responsible mechanisms; and assess if NKT cells stimulate fibrogenesis. NKT cells were analyzed in wildtype mice and Patched-deficient (Ptc(+/-)) mice with an overly active Hedgehog (Hh) pathway, before and after feeding methionine choline-deficient (MCD) diets to induce NASH-related fibrosis. Effects of NKT cell-derived factors on hepatic stellate cells (HSC) were examined and fibrogenesis was evaluated in CD1d-deficient mice that lack NKT cells. NKT cells were quantified in human cirrhotic and nondiseased livers. During NASH-related fibrogenesis in wildtype mice, Hh pathway activation occurred, leading to induction of factors that promoted NKT cell recruitment, retention, and viability, plus liver enrichment with NKT cells. Ptc(+/-) mice accumulated more NKT cells and developed worse liver fibrosis; CD1d-deficient mice that lack NKT cells were protected from fibrosis. NKT cell-conditioned medium stimulated HSC to become myofibroblastic. Liver explants were 2-fold enriched with NKT cells in patients with non-NASH cirrhosis, and 4-fold enriched in patients with NASH cirrhosis.Hh pathway activation leads to hepatic enrichment with NKT cells that contribute to fibrosis progression in NASH.

Chemokines direct leukocyte trafficking and positioning within tissues. They thus play critical roles in regulating immune responses and inflammation. The chemokine system is complex involving interactions between multiple chemokines and their receptors that operate in combinatorial cascades with adhesion molecules. The involvement of multiple chemokines and chemokine receptors in these processes brings flexibility and specificity to recruitment. The hepatic vascular bed is a unique low flow environment through which leukocyte are recruited to the liver during homeostatic immune surveillance and in response to infection or injury. The rate of leukocyte recruitment and the nature of cells recruited through the sinusoids in response to inflammatory signals will shape the severity of disease. At one end of the spectrum fulminant liver failure results from a rapid recruitment of leukocytes that leads to hepatocyte destruction and liver failure at the other diseases such as chronic hepatitis C infection may progress over many years from hepatitis to fibrosis and cirrhosis. Chronic hepatitis is charactezised by a T lymphocyte rich infiltrate and the nature and outcome of hepatitis will depend on the T cell subsets recruited, their activation and function within the liver. Different subsets of effector T cells have been described based on their secretion of cytokines and specific functions. These include Th1 and Th2 cells and more recently Th17 and Th9 cells which are associated with different types of immune response and which express distinct patterns of chemokine receptors that promote their recruitment under particular conditions. The effector function of these cells is balanced by the recruitment of regulatory T cells that are able to suppress antigen-specific effectors to allow resolution of immune responses and restoration of immune homeostasis. Understanding the signals that are responsible for recruiting different lymphocyte subsets to the liver will elucidate disease pathogenesis and open up new therapeutic approaches to modulate recruitment in favour of resolution rather than injury.

Chemokines direct leukocyte trafficking and positioning within tissues, thus playing critical roles in regulating immune responses and inflammation. The chemokine system is complex, involving interactions between multiple chemokines and their receptors that operate in combinatorial cascades with adhesion molecules. The involvement of multiple chemokines and chemokine receptors in these processes brings flexibility and specificity to recruitment. The hepatic vascular bed is a unique low-flow environment through which leukocytes are recruited to the liver during homeostatic immune surveillance and in response to infection or injury. The rate of leukocyte recruitment and the nature of cells recruited through the sinusoids in response to inflammatory signals will shape the severity of disease. At one end of the spectrum, fulminant liver failure results from a rapid recruitment of leukocytes that leads to hepatocyte destruction and liver failure; at the other end, diseases such as chronic hepatitis C infection may progress over many years from hepatitis to fibrosis and cirrhosis. Chronic hepatitis is characterized by a T lymphocyte-rich infiltrate and the nature and outcome of hepatitis will depend on the T cell subsets recruited, their activation and function within the liver. Different subsets of effector T cells have been described based on their secretion of cytokines and specific functions. These include Th1 and Th2 cells, and more recently Th17 and Th9 cells, which are associated with different types of immune response and which express distinct patterns of chemokine receptors that promote their recruitment under particular conditions. The effector function of these cells is balanced by the recruitment of regulatory T cells that are able to suppress antigen-specific effectors to allow resolution of immune responses and restoration of immune homeostasis. Understanding the signals that are responsible for recruiting different lymphocyte subsets to the liver will elucidate disease pathogenesis and open up new therapeutic approaches to modulate recruitment in favor of resolution rather than injury.

Background & Aims Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that requires long-term immunosuppressive therapy. Although most patients have an excellent response to standard therapy (azathioprine in combination with corticosteroids), approximately 10%–15% have intolerance or an insufficient response to azathioprine treatment. We investigated whether 6-mercaptopurine (6-MP) is an effective second-line therapy for patients with AIH. Methods We performed a retrospective study of 22 patients with AIH who were switched to 6-MP therapy after treatment with the combination of azathioprine and prednisolone at 2 tertiary care institutions in Europe (Germany and the United Kingdom) before November 15, 2014. We performed statistical analyses of data on clinical and biochemical responses collected 4 weeks after 6-MP treatment and then at regular physician visits. Results A total of 15 of 20 patients with prior azathioprine intolerance (75%) responded to 6-MP treatment; 8 of these patients had a complete response and 7 had partial remission, based on biochemical features. In these 15 patients, 6-MP was well tolerated, whereas the 5 remaining patients had to be switched to different immunosuppressive regimes because of 6-MP intolerance. The 2 patients with insufficient response to azathioprine treatment also showed no response to 6-MP. Conclusions In patients with AIH and azathioprine intolerance, 6-MP seems to be an effective and well-tolerated second-line treatment. 6-MP might be ineffective in patients with insufficient response to azathioprine.

Our objective was to determine the prevalence and risk factors for HIV infection among female sex workers in Johannesburg, South Africa. A cross-sectional survey of female sex workers was conducted using interviewer-administered questionnaires. Prevalent sexually transmitted infections including HIV were evaluated through standard laboratory testing. HIV infection was identified in 137 (46.4%) of 295 subjects tested. Increasing frequency of condom use was significantly negatively associated with HIV infection (odds ratio [OR] for moderate use = 0.21; 95% confidence interval [CI]: [0.09, 0.50]; OR for high use = 0.14; 95% CI: [0.06, 0.34]). Sex workers aged ≥29 years reported significantly different patterns of behaviour than younger workers. Among women aged ≥29, a negative association with HIV infection (OR = 0.16; 95% CI: [0.07, 0.38]) was found, but only among those not infected with Neisseria gonorrhoeae. Older women in the Johannesburg sex industry may have adaptive behavioural strategies besides condom usage which reduce their risk of acquiring HIV. However, older sex workers with gonorrhoea constitute a high-risk subgroup.

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]. 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]. 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]. It is thus logical to look for defective Treg function in autoimmune hepatitis, an archetypal autoimmune disease [4]. Although previous studies reported reduced numbers and defective Treg function in AIH [5–7], 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]. They also quantified Treg within the liver and confirmed previous findings that intrahepatic Treg frequencies mirror those

Autoimmune hepatitis (AIH) is characterized by a T-cell rich infiltrate associated with lobular and interface hepatitis, hypergammaglobulinemia and production of autoantibodies. Genetic risk is linked to the HLA particularly DRB1*0301 and DRB1*0401 alleles in North American and European Caucasian populations. It has recently been suggested that functional deficiencies in CD4+CD25+CD127lowFOXP3+ regulatory T cells contribute to the breakdown of immune tolerance that results in AIH. Most patients respond to immunosuppressive therapy with corticosteroids and can be maintained in remission by low-dose corticosteroid treatment and/or azathioprine. For those who progress to end-stage disease liver transplantation is an effective treatment although it is associated with recurrence. In the future it is likely that biological therapies will allow more targeted therapy designed to switch the balance to immune regulation and thereby restore immune homeostasis. Although treatment for many cases is relatively straightforward and successful problems are encountered in those who fail to respond to standard treatment, are unable to tolerate it or relapse. In such cases alternative therapies should be considered. In addition treatment is complicated in some patients by comorbidity and special care is required during and after pregnancy. We will discuss the current and future therapeutic options for patients with difficult to treat AIH.

Innate immune system is the first line of defence against invading pathogens that is critical for the overall survival of the host. Human liver is characterised by a dual blood supply, with 80% of blood entering through the portal vein carrying nutrients and bacterial endotoxin from the gastrointestinal tract. The liver is thus constantly exposed to antigenic loads. Therefore, pathogenic microorganism must be efficiently eliminated whilst harmless antigens derived from the gastrointestinal tract need to be tolerized in the liver. In order to achieve this, the liver innate immune system is equipped with multiple cellular components; monocytes, macrophages, granulocytes, natural killer cells, and dendritic cells which coordinate to exert tolerogenic environment at the same time detect, respond, and eliminate invading pathogens, infected or transformed self to mount immunity. This paper will discuss the innate immune cells that take part in human liver inflammation, and their roles in both resolution of inflammation and tissue repair.

Introduction Progressive steatohepatitis is characterised by increased inflammatory cell infiltration. Osteopontin (OPN) is a cytokine intricately associated with immune-cell accumulation, and we reported that NKT cells accumulate to promote hepatic injury in NASH. We hypothesise that OPN promotes NASH progression by supporting NKT migration across hepatic sinusoidal endothelium. Methods Mice were fed chow or methionine-choline deficient (MCD) diet to induce NASH. After 4 weeks, mice were sacrificed; severity of disease assessed by serum aminotransferase (AST), liver OPN quantified by QRTPCR and immunohistochemistry, blood OPN measured by ELISA. In separate studies, MCD-fed mice were treated with sham or OPN aptamers, and FACS used to quantify liver NKTs. Primary human hepatic sinusoidal endothelial cells (HSEC) were stimulated with recombinant (r)OPN (0–1000 ng/ml), with or without TNFa (20 ng/ml)+IFNg (100 ng/ml), and expression of adhesion molecules (ICAM1, VCAM1) and chemokines (CXCL9, 10, 11, 16) assessed. To assess lymphocyte migration, lymphocytes were perfused over rOPN—or vehicle-treated-HSEC, with or without TNFa+IFNg. In separate experiments, TNFa+IFNg stimulated-HSEC were treated with OPN aptamers or blocking antibodies, and total lymphocyte adhesion recorded. Human NASH livers were immunostained for OPN, plasma measured for OPN, and liver NKT numbers from normal or NASH-cirrhotic patients quantified by FACS. Results In mice, MCD-induced NASH upregulated expression of liver OPN by threefolds (p v b 3 , ICAM1, VCAM1, and CXCL16. In humans, liver and plasma OPN was significantly upregulated in NASH; livers from NASH-cirrhosis harboured twofolds more CD4 and threefolds more NKT cells (p Conclusion Liver and plasma OPN levels are upregulated during steatohepatitis in mice and humans, and promote liver NKT accumulation. OPN neutralisation significantly reduces lymphocyte subset recruitment and liver injury, suggesting that OPN is a promising anti-inflammatory target in steatohepatitis. Competing interests None declared.

INTRODUCTION: In patients with autoimmune hepatitis, efficient immunosuppressive therapy is essential to avoid progression to cirrhosis. There is no established second line therapy for patients failing standard therapy with steroids and azathioprine. The aim of this study was to examine the possible role of mycophenolate mofetil (MMF) as second line treatment of autoimmune hepatitis (AIH). PATIENTS We were able to identify 37 patients (29 women, 8 men) with AIH proven according to AND METHODS: International AIH Group criteria who failed standard therapy. One patient on MMF was excluded due to non-compliance. A total of 28 of 36 patients had experienced side effects necessitating stop of treatment. One patient stopped azathioprine due to pregnancy. A total of nine patients did not respond sufficiently to azathioprine. A total of four patients with a treatment duration of 3 months or less because of severe side effects were considered as intolerant to MMF. Remission was defined as aspartate transaminase (ASP) < twice upper normal limit (UNL). RESULTS: Of 36 patients on MMF included in the analysis, 14 patients (39%) experienced remission. A total of 22 patients (61%) did not respond sufficiently to MMF. The response rate to MMF was dependent on the cause of treatment cessation of azathioprine. Of eight patients with prior nonresponse to azathioprine, six (75%) did not respond to MMF and only two (25%) reached biochemical remission. Of 28 patients with azathioprine intolerance in 16 (57%) patients, the response to MMF was insufficient and in 12 patients (43%) remission was reached. The difference did not reach statistical significance due to the relatively small numbers included. CONCLUSION: In the light of its good tolerability, MMF seems to be an alternative for patients who could not tolerate azathioprine previously. However, our data suggest that a majority of patients fail MMF particularly if they are switched because of an insufficient response to azathioprine.

Hepatitis C virus (HCV)-induced cirrhosis is the most common indication for liver transplantation (LT). However, graft reinfection is nearly universal. The choice of immunosuppression, including the calcineurin inhibitor (CNI), may have some effect on severity of recurrence and graft survival. In addition, HCV recurrence may have some impact on metabolism of immunosuppressive drugs. In this retrospective study, we examined the dose and blood levels of tacrolimus (TAC) and cyclosporin A (CYA) in HCV patients consecutively undergoing transplantation (TAC, n = 44; CYA, n = 60) and surviving 12 months post-LT. In addition, we examined the CNI dose and blood levels in an age- and gender-matched comparison group of patients who were transplanted for alcoholic liver disease (ALD) (TAC, n = 44; CYA, n = 47). During the 12-month period of observation, TAC levels were significantly higher for HCV than for ALD patients (P = 0.002). The dose of TAC decreased over time for both HCV and ALD patients (P < 0.001), but the reduction was greater for HCV patients (P = 0.03). CYA dose decreased over time for both groups (P < 0.001) but a greater reduction was observed for the HCV group (P = 0.007). For both HCV and ALD patients, CYA levels decreased over time (P < 0.001) but there was no significant difference between HCV and ALD patients. Thus, to maintain comparable blood levels, a greater reduction of dose was required for HCV than for ALD patients. In conclusion, our observations demonstrate a likely effect of HCV infection on CNI metabolism, an effect that is not clearly due to graft damage. Physicians need to be alert to this interaction and to the need to respond quickly to changes in CNI levels that may be associated with HCV infection and with HCV clearance during antiviral therapy.