Rodrigo Liberal

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)

Inhibition of Interleukin-17 Promotes Differentiation of CD25– Cells Into Stable T Regulatory Cells in Patients With Autoimmune Hepatitis

BACKGROUND & AIMS Patients with autoimmune hepatitis (AIH) have reduced numbers and function of CD4+CD25(high)FOXP3+ T regulatory cells (Tregs). Tregs can be generated from CD25⁻ (ngTreg) cells, which suppress the immune response less efficiently than Tregs. We investigated whether their differentiation into T-helper (Th)17 cells, an effector subset that has the same CD4+ progenitors as Tregs, accounts for the reduced suppressive functions of ngTregs. We investigated whether blocking interleukin (IL)-17 increased the immunosuppressive activity of Tregs. METHODS ngTregs were generated from 36 patients with AIH and 23 healthy subjects (controls). During Treg differentiation, expression of IL-17 was inhibited by physical removal of IL-17-secreting cells, exposure to recombinant transforming growth factor β or neutralizing antibodies against IL-6 and IL-1β (to promote differentiation of ngTregs vs Th17 cells), small inhibitory RNAs specific for the Th17 transcription factor RORC, or a combination of all these approaches. RESULTS ngTregs from patients with AIH contained greater proportions of IL-17+ and RORC+ cells than Tregs from controls. All approaches to inhibit IL-17 increased expression of FOXP3 by ngTregs and their suppressive functions. Inhibition of IL-17 led to development of ngTregs that were phenotypically stable and did not acquire proinflammatory properties after exposure to IL-6 and IL-1β. CONCLUSIONS Blocking Th17 allows ngTregs to differentiate into functionally stable immune inhibitory cells; this approach might be developed for therapy of patients with AIH.

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)

Pathogenesis of autoimmune hepatitis

The mechanisms underlying the pathogenesis of autoimmune hepatitis are not fully understood, though there is growing evidence that genetic predisposition, molecular mimicry and/or impairment of regulatory T-cells are involved in the initiation and perpetuation of the autoimmune liver attack. The histological picture of interface hepatitis, characterized by a dense portal mononuclear cell infiltrate, was the first to suggest an autoaggressive cellular immune attack in the pathogenesis of this condition. Liver damage is likely to be orchestrated by CD4(pos) T-cells recognizing an autoantigenic liver peptide. For autoimmunity to arise, the peptide must be presented by antigen-presenting cells to naïve CD4(pos) T-helper (Th0) cells. Once activated, Th0-cells can differentiate into Th1-, Th2-, or Th17-cells, initiating a cascade of immune reactions that are determined by the cytokines they produce. Autoantigen recognition and the above effector mechanisms are opposed by regulatory T-cells, a cell subset numerically and functionally impaired in autoimmune hepatitis.

Autoimmune hepatitis: Contrasts and comparisons in children and adults - a comprehensive review.

This review concentrates on autoimmune hepatitis (AIH), a liver disorder affecting both children and adults, characterized by inflammatory liver histology, elevated transaminase levels, circulating non-organ-specific autoantibodies, and increased levels of immunoglobulin G, in the absence of a known aetiology. Two types of AIH are recognized according to seropositivity: smooth muscle antibody and/or antinuclear antibody define AIH type 1, while antibodies to liver-kidney microsome type 1 and/or liver cytosol type 1 define AIH type 2. AIH type 1 affects both adults and children, while AIH type 2 is mainly a paediatric disease, though it does occasionally affects young adults. There is a female predominance in both types. AIH is particularly aggressive in children/adolescents, progressing rapidly unless immunosuppressive treatment is started promptly. With appropriate treatment 80% of patients achieve remission and long-term survival. In childhood/adolescence, sclerosing cholangitis with strong autoimmune features, including interface hepatitis and serological features identical to AIH type 1, is as prevalent as AIH, but it affects boys and girls equally. The differential diagnosis relies on cholangiographic studies. In autoimmune sclerosing cholangitis, liver parenchymal damage responds satisfactorily to immunosuppressive treatment, whereas bile duct disease progresses in 50% of cases, leading to a worse prognosis and higher transplantation requirement; it has a higher recurrence rate after transplant than AIH. AIH can arise de novo in patients transplanted for non-autoimmune liver disease. Post transplant de novo AIH affects children and adults and responds well to the same treatment schedule used for classical AIH, but not to that used for acute rejection.

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.

Liver transplantation and autoimmune liver diseases

Liver transplantation (LT) is an effective treatment for patients with end‐stage autoimmune liver diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Indications for LT for these diseases do not differ substantially from those used for other acute or chronic liver diseases. Despite the good outcomes reported, the recurrence of autoimmune liver disease is relatively common in the allograft. In addition, it has become apparent that autoimmunity and autoimmune liver disease can arise de novo after transplantation for nonautoimmune liver disorders. An awareness of the existence of recurrent autoimmune liver diseases and de novo autoimmune hepatitis after LT has important clinical implications because their management differs from the standard antirejection treatment and is similar to the management of classic autoimmune liver diseases in the native liver. Liver Transpl 19:1065‐1077, 2013.