Charlotte R. Grant

Regulatory T-cells in autoimmune diseases: Challenges, controversies and—yet—unanswered questions

Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.

Autoimmune hepatitis: a comprehensive review.

Autoimmune hepatitis (AIH) is an immune-mediated liver disorder characterised by female preponderance, elevated transaminase and immunoglobulin G levels, seropositivity for autoantibodies and interface hepatitis. Presentation is highly variable, therefore AIH should be considered during the diagnostic workup of any increase in liver enzyme levels. Overlap/variant forms of the disease, presenting with concomitant features of primary biliary cirrhosis or primary sclerosing cholangitis are increasingly recognised. AIH is exquisitely responsive to immunosuppressive treatment, which should be instituted promptly to prevent rapid deterioration and promote remission and long-term survival. Difficult-to-treat or non-responsive patients should be treated with mycophenolate mofetil or, failing that, calcineurin inhibitors. Persistent failure to respond or lack of adherence to treatment result in end-stage liver disease. These patients, and those with fulminant liver failure (encephalopathy grade II-IV) at diagnosis, will require liver transplantation. The pathogenesis of AIH is not fully understood, although there is mounting evidence that genetic susceptibility, molecular mimicry and impaired immunoregulatory networks contribute to the initiation and perpetuation of the autoimmune attack. Liver damage is thought to be mediated primarily by CD4(pos) T-cells, although recent studies support the involvement of diverse populations, including Th17 cells. Animal models faithfully representing the human condition are needed to unravel the contribution of innate and adaptive, effector and regulatory immune responses. A deeper understanding of the pathogenesis of AIH is likely to contribute to the development of novel treatments, such as the adoptive transfer of autologous expanded antigen-specific regulatory T-cells, which ultimately aim to restore tolerance to liver-derived antigens.

Dysfunctional CD39(POS) Regulatory T Cells and Aberrant Control of T-Helper Type 17 Cells in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T‐cell (Treg) impairments. Tregs express CD39, an ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39pos Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39pos Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg subsets to suppress both proliferation of effector T cells and interleukin (IL)‐17 production was evaluated. In AIH, CD39pos Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL‐17 production by effector CD4 T cells. Moreover, these CD39pos Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon‐gamma or IL‐17 upon challenge with proinflammatory stimuli. Conclusions: In AIH, CD39pos Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL‐17 production by effector CD4 T cells. CD39pos Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells. (Hepatology 2014;59:1007–1015)

The impaired immune regulation of autoimmune hepatitis is linked to a defective galectin‐9/tim‐3 pathway

In autoimmune hepatitis (AIH), liver‐damaging CD4 T cell responses are associated with defective CD4posCD25pos regulatory T cells (T‐regs). Galectin‐9 (Gal9), a β‐galactosidase–binding protein expressed by T‐regs, is key to their function, inhibiting T helper 1 immune responses by binding T cell immunoglobulin and mucin domain 3 (Tim‐3) on CD4 effector cells. We investigated whether impaired immunoregulation in AIH results from reduced expression of Gal9 in T‐regs and/or Tim‐3 on CD4 effector cells. Circulating Gal9posCD4posCD25pos and Tim‐3posCD4posCD25neg T cell phenotype was assessed by flow cytometry in 75 AIH patients. To evaluate whether Tim‐3 expression renders CD4posCD25neg T cells amenable to T‐reg control, purified CD4posCD25negTim‐3pos (Tim‐3pos) and CD4posCD25negTim‐3neg (Tim‐3neg) cells were cocultured with T‐regs. To determine whether Gal9 expression is essential to function, T‐regs were treated with small interfering RNA (siRNA) to repress Gal‐9 translation; T‐reg suppressor function was assessed by proliferation. In AIH, Tim‐3pos cells within CD4posCD25neg cells and their T‐betpos and RORCpos subsets were fewer and contained higher numbers of interferon‐γ (IFNγ)pos and interleukin (IL)‐17pos cells than healthy subjects (HS). In AIH and HS, Tim‐3pos cells proliferated less vigorously and were more susceptible to T‐reg control than Tim‐3neg cells. In AIH, Gal9posT‐regs were fewer and contained less FOXP3pos, IL‐10pos, and transforming growth factor βpos and more IFNγpos and IL‐17pos cells than HS. siRNA treatment of Gal‐9pos T‐regs drastically reduced T‐reg ability to suppress CD4posCD25neg and Tim‐3pos cell proliferation in AIH and HS. Tim‐3pos cell percentage correlated inversely with aminotransferase and CD25negT‐betpos cell values. Conclusion: Reduced levels of Tim‐3 on CD4posCD25neg effector cells and of Gal9 in T‐regs contribute to impaired immunoregulation in AIH by rendering effector cells less prone to T‐reg control and T‐regs less capable of suppressing. (HEPATOLOGY 2012)

Diagnostic criteria of autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic immune-mediated liver disorder characterised by female preponderance, elevated transaminase and immunoglobulin G levels, seropositivity for autoantibodies and interface hepatitis. Presentation is highly variable, therefore AIH should be considered during the diagnostic workup of any increase in liver enzyme levels. A set of inclusion and exclusion criteria for the diagnosis of AIH have been established by the International Autoimmune Hepatitis Group (IAIHG). There are two main types of AIH: type 1, positive for anti-nuclear (ANA) and/or anti-smooth muscle antibodies (SMAs) and type 2, defined by the presence of anti-liver kidney microsomal antibody type 1 (LKM-1) and/or anti-liver cytosol type 1 (LC-1) autoantibodies. The central role of autoantibodies in the diagnosis of AIH has led the IAIHG to produce a consensus statement detailing appropriate and effective methods for their detection. Autoantibodies should be tested by indirect immunofluorescence at an initial dilution of 1/40 in adults and 1/10 in children on a freshly prepared rodent substrate that includes kidney, liver and stomach sections to allow for the simultaneous detection of all reactivities relevant to AIH. Anti-LKM-1 is often confused with anti-mitochondrial antibody (AMA) if rodent kidney is used as the sole immunofluorescence substrate. The identification of the molecular targets of anti-LKM-1 and AMA has led to the establishment of immuno-assays based on the use of the recombinant or purified autoantigens. Perinuclear anti-nuclear neutrophil antibody (p-ANNA) is an additional marker of AIH-1; anti soluble liver antigen (SLA) antibodies are specific for autoimmune liver disease, can be present in AIH-1 and AIH-2 and are associated with a more severe clinical course. Anti-SLA are detectable by ELISA or radio-immuno-assays, but not by immunofluorescence. AIH is exquisitely responsive to immunosuppressive treatment, which should be instituted promptly to prevent rapid deterioration and promote remission and long-term survival.

Autoimmune hepatitis after liver transplantation.

Liver transplantation is an effective treatment for patients with end-stage acute and chronic autoimmune hepatitis. However, despite the good outcomes reported, disease recurrence is relatively common in the allograft. In addition, autoimmunity and autoimmune liver disease can arise de novo after transplantation for non-autoimmune liver disorders. Little is known about the mechanisms by which autoimmune diseases develop after liver transplantation, but genetic factors, molecular mimicry, impaired regulatory T-cell responses, and exposures to new alloantigens might be involved. Regardless of the pathogenic mechanisms, it is important to remain aware of the existence of recurrent and de novo autoimmune hepatitis after liver transplantation; these disorders are similar to classic autoimmune hepatitis and are therefore not treated with standard antirejection strategies.

In autoimmune hepatitis type 1 or the autoimmune hepatitis–sclerosing cholangitis variant defective regulatory T‐cell responsiveness to IL‐2 results in low IL‐10 production and impaired suppression

Defective immune regulation plays a permissive role enabling effector cells to initiate and perpetuate tissue damage, eventually resulting in autoimmune disease. Numerical and functional regulatory T‐cell (Treg) impairment has been previously reported in autoimmune liver disease (AILD; including autoimmune hepatitis and autoimmune sclerosing cholangitis ASC). However, in these early reports, Tregs were phenotypically defined as CD4+CD25+ or CD4+CD25high cells. In the current study, we reexamined phenotypic and functional properties of Tregs by adopting a more refined definition of these cells that also includes negativity or low level of expression of CD127. We studied 43 AILD patients and 22 healthy subjects (HSs) and found that CD4+CD25+CD127− Tregs were decreased in the former. This decrease was more marked in patients with active disease than in those in remission. In AILD, Treg frequencies correlated inversely with parameters of disease activity and were not affected by immunosuppressive treatment. We also document, for the first time, that, in AILD, bona‐fide Tregs produce less interleukin (IL)−10 and are impaired in their ability to suppress CD4+CD25− target cell proliferation, a feature that in HSs, but not in AILDs, is dependent, at least in part, on IL‐10 secretion. Decreased IL‐10 production by Tregs in AILD is linked to poor responsiveness to IL‐2 and phospho signal transducer and activator of transcription 5 up‐regulation. Conclusion: Tregs are numerically impaired in AILD, this impairment being more prominent during active disease. Notably, defective IL‐10 production, resulting from low Treg responsiveness to IL‐2, contributes to Treg functional impairment. (Hepatology 2015;62:863–875)

T-regs in autoimmune hepatitis-systemic lupus erythematosus/mixed connective tissue disease overlap syndrome are functionally defective and display a Th1 cytokine profile

Autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinaemia, autoantibodies and interface hepatitis, is occasionally associated with systemic autoimmune manifestations [systemic lupus erythematosus (SLE); mixed connective tissue disease (MCTD)]. In both AIH and SLE/MCTD numerical and/or functional impairment of regulatory T-cells (T-regs) is believed to favour autoimmunity. To investigate whether immune-tolerance breakdown profiles differ in patients with AIH and SLE/MCTD, isolated AIH or systemic autoimmunity, we studied phenotypic and functional features of T-regs in 10 patients with AIH-SLE/MCTD, 22 with AIH, 12 with SLE and 20 healthy subjects. Compared to health, CD4(pos)CD25(pos) cells were decreased in number and expressed high levels of the CD127 activation marker in all three disease groups; in AIH-SLE/MCTD and in SLE they displayed low levels of FOXP3. In AIH-SLE/MCTD, they also contained a high proportion of IFNγ positive cells, indicating a Th1 profile. Similarly, in AIH-SLE/MCTD, CD4(pos)CD25(pos)CD25(high) T-regs were reduced in number and contained an increased proportion of activated CD127(pos) and IFNγ(pos) cells. Such skewing towards a Th1 profile was also present at effector level, as a high frequency of IFNγ-producing cells was observed within AIH-SLE/MCTD CD4(pos)CD25(neg) responder cells. Impairment in suppressor function both of CD4(pos)CD25(pos) cells and CD4(pos)CD25(pos)CD127(neg) T-regs was observed in all three autoimmune conditions, but while addition of CD4(pos)CD25(pos)CD127(neg) T-regs decreased CD4(pos)CD25(neg) responder cell proliferation in healthy subjects and partially in AIH patients, it had no effect in AIH-SLE/MCTD and SLE patients. In conclusion, in AIH-SLE/MCTD T-regs display a distinctive phenotypic and functional signature, characterized by marked activation, elevated IFNγ production and by a profound impairment of suppressive function, suggesting that multiple autoimmune manifestations may derive from a complex defect of immune-regulation.

Regulatory T cells: Mechanisms of suppression and impairment in autoimmune liver disease

There are three classic liver diseases with probable autoimmune etiology: primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. The occurrence of these autoimmune conditions is determined by the breakdown of immune‐regulatory mechanisms that in health are responsible for maintaining immunological tolerance against self‐antigens. Among the multiple T cell subsets with suppressive function, the regulatory T cells (Tregs), defined by the expression of CD4, the IL‐2 receptor α chain (CD25), and the transcription factor FOXP3, have emerged as having a central role in maintaining immune‐tolerance to autoantigens. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways, and the production of anti‐inflammatory cytokines. In all the three autoimmune liver diseases mentioned above, there is evidence pointing for either a reduced frequency and/or function of Tregs. Here, we review the definition, phenotypic characteristics, and mechanisms of suppression employed by Tregs and then we discuss the evidence available pointing to their impairment in patients with autoimmune liver disease.

Diagnostic and clinical significance of anti-centromere antibodies in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver characterised by biochemical evidence of cholestasis, elevated alkaline phosphatase levels and the presence of the highly disease specific anti-mitochondrial autoantibodies. Extra-hepatic autoimmune manifestations are common, including rheumatic disorders, such as systemic sclerosis (SSc). Notably, PBC is the most frequent autoimmune liver disease in SSc patients. Based on skin lesion extension, two major SSc disease subgroups are recognised: limited cutaneous SSc (lSSc) and diffuse cutaneous SSc. Anti-centromere antibody (ACA) positivity is highly characteristic of SSc, with up to 90% prevalence in lSSc patients. ACA has also been found in up to 30% of PBC patients and 80% of patients with a PBC/SSc overlap syndrome. The diagnostic and clinical significance of ACA positivity in patients with PBC without SSc has recently been under investigation, with several studies highlighting links to severe bile duct injury and portal hypertension. This review discusses the diagnostic and clinical relevance of ACA in patients with PBC, with or without SSc.