Maria Serena Longhi

Functional Study of CD4+CD25+ Regulatory T Cells in Health and Autoimmune Hepatitis

Regulatory CD4+CD25+ T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-γ production by CD4+CD25− and CD8+ T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-β, suggesting a mechanism of linked immunosuppression. Tregs/CD4+CD25− T cell cocultures lead to similar changes in IFN-γ and IL-10 secretion in patients and controls, whereas increased TGF-β secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8+ T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4+CD25− T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4+CD25− T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4+CD25− T cell spontaneous apoptosis may contribute to the development of autoimmunity.

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.

Aetiopathogenesis of autoimmune hepatitis

Interface hepatitis, histological hallmark of autoimmune hepatitis (AIH), is a dense portal mononuclear cell infiltrate invading the parenchyma, consisting of CD4 and CD8 T-lymphocytes, monocytes/macrophages and plasma cells. What triggers AIH is unknown, but there is evidence that failure of immune homeostasis plays a permissive role allowing liver autoantigen-specific cells to attack hepatocytes. Damage is likely to be orchestrated by CD4 T-lymphocytes recognizing an autoantigenic liver peptide. For autoimmunity to arise, the peptide, embraced by an HLA class II molecule, must be presented to naïve CD4 T-helper (T(H)0) cells by professional antigen-presenting cells. Once activated, T(H)0 cells can differentiate into T(H)1 cells, which are pivotal to macrophage activation, enhance HLA class I expression, rendering liver cells vulnerable to CD8 T-cell attack, and induce HLA class II expression on hepatocytes; or into T(H)2 cells producing IL-4, IL-10 and IL-13, cytokines favouring autoantibody production by B-lymphocytes. Autoantigen recognition is tightly controlled by regulatory mechanisms, such as those exerted by CD4+CD25+ regulatory T-cells (T-regs). Numerical and functional T-reg impairment characterizes AIH, particularly during active disease. Advances in the study of autoreactive T-cells stem mostly from AIH type 2, where the main autoantigen, CYP2D6, is known enabling characterization of antigen-specific immune responses. Monocyte involvement in the autoimmune liver attack has been recently reported.

Reactivation of replication of hepatitis B and C viruses after immunosuppressive therapy: an unresolved issue

The liver is susceptible to the toxic effects of many cytotoxic or immunosuppressive treatments. However, in carriers of hepatitis B virus (HBV) and, less frequently, of hepatitis C virus, liver damage due to reactivation of viral replication can occur after withdrawal of immunosuppressive drugs. These reactivations, which are associated with fulminant forms of hepatitis in up to 25% of cases, are observed both in symptom-free chronic carriers of hepatitis B surface antigen and in patients who have chronic hepatitis B or C and concurrent haematological tumours or solid neoplasms or who have received transplants. HBV-related complications may cause delays or modifications of therapy, and the chance of cure is reduced. In this review, we analyse clinical, biochemical, and serological issues in reactivation of viral replication and examine the role of immune reactions in the pathogenesis and the possible toxicity of immunosuppressive drugs. We emphasise the importance of identifying predictive markers of a clinically relevant reactivation, review difficulties in drug prevention and treatment, indicate studies that are needed to address the key clinical issues, and give practical recommendations to practising physicians and oncologists.

A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis

Immunotolerance is maintained by regulatory T cells (Tregs), including CD4+CD25hi, CD8+CD28−, γδ, and CD3+CD56+ [natural killer T (NKT)] cells. CD4+CD25hi cells are impaired in children with autoimmune hepatitis (AIH). Little is known about Tregs in adults with AIH. The aim of this study was to investigate the frequency and function of Treg subsets in adult patients with AIH during periods of active disease and remission. Forty‐seven AIH patients (16 with active disease and 31 in remission) and 28 healthy controls were studied. Flow cytometry was used to evaluate surface markers and function‐related intracellular molecules in γδ, CD8+CD28−, NKT, and CD4+CD25hi cells. CD4+CD25hi T cell function was determined by the ability to suppress proliferation and interferon gamma (IFN‐γ) production by CD4+CD25− target cells. Liver forkhead box P3–positive (FOXP3+) cells were sought by immunohistochemistry. In AIH patients, particularly during active disease, CD4+CD25hi T cells were fewer, expressed lower levels of FOXP3, and were less effective at inhibiting target cell proliferation versus healthy controls. Moreover, although the numbers of CD8+CD28− T cells were similar in AIH patients and healthy controls, NKT cells were numerically reduced, especially during active disease, and produced lower quantities of the immunoregulatory cytokine interleukin‐4 versus controls. In contrast, γδ T cells in AIH patients were more numerous versus healthy controls and had an inverted Vδ1/Vδ2 ratio and higher IFN‐γ and granzyme B production; the latter was correlated to biochemical indices of liver damage. There were few FOXP3+ cells within the portal tract inflammatory infiltrate. Conclusion: Our data show that the defect in immunoregulation in adult AIH is complex, and γδ T cells are likely to be effectors of liver damage. (HEPATOLOGY 2010)

Expansion and de novo generation of potentially therapeutic regulatory T cells in patients with autoimmune hepatitis

CD4+CD25+ regulatory T cells (T‐regs) are central to the maintenance of immune tolerance and represent an immune intervention candidate in autoimmune hepatitis (AIH), a condition characterized by impaired T‐reg number and function. We investigated whether T‐regs can be expanded from the existing CD4+CD25+ T cell pool and generated de novo from CD4+CD25− T cells in AIH patients and healthy controls. Purified CD4+CD25+ and CD4+CD25− T cells from 24 patients with type 1 AIH and 22 healthy controls were cultured for up to 5 weeks with anti‐CD3/anti‐CD28 T cell expander and high‐dose interleukin‐2 (IL‐2). Cell phenotypes, suppressor ability, forkhead winged/helix transcription factor box P3 (FOXP3) gene, and protein expression were assessed weekly by cytofluorimetry, proliferation assay, real‐time polymerase chain reaction (PCR), and immunoblot. During culture, the number of CD4+CD25+ T cells derived from the existing T‐reg pool (expanded T‐regs) and generated de novo from CD4+CD25− T cells (newly generated T‐regs) increased constantly up to week 4 in both healthy controls and, to a lesser extent, in AIH patients. Expanded T‐regs retained conventional T‐reg phenotype and, compared with baseline, demonstrated more vigorous suppressive function and increased FOXP3 gene and protein expression. Newly generated T‐regs not only acquired T‐reg phenotype but underwent greater growth and were more resistant to apoptosis than expanded T‐regs. Their suppressive function augmented throughout culture, reaching a peak at week 4, preceded by a peak FOXP3 gene and protein expression at week 2. Suppressor function and FOXP3 expression of both expanded and newly generated T‐regs were higher in normal controls than in AIH patients. Conclusion: Functionally enhanced T‐regs can be expanded and generated de novo in patients with AIH. This finding may assist in reconstituting impaired immune regulation and restoring peripheral tolerance through T‐reg infusion in this condition. (HEPATOLOGY 2008;47:581–591.)

Cytochrome P450IID6-specific CD8 T cell immune responses mirror disease activity in autoimmune hepatitis type 2†

Autoimmune hepatitis type 2 (AIH‐2) is a severe organ‐specific disorder characterized by liver kidney microsomal antibody type 1 targeting cytochrome P4502D6 (CYP2D6). Growing evidence implicates the involvement of CD8 T cell immune responses in its pathogenesis. We investigated CYP2D6‐specific CD8 T cell human leukocyte antigen (HLA)‐A2 restricted responses in AIH‐2 (20 patients, 11 HLA‐A2+). Binding affinity of CYP2D6 peptides to HLA‐A2 was predicted by the algorithm SYFPEITHI and assessed in vivo by T2 cell assays. CD8 T cell interferon (IFN)‐γ production was assessed via intracellular cytokine staining, cytotoxicity via chromium release assay, and frequency of circulating and intrahepatic CYP2D6‐specific CD8 T cells via tetramer staining. CYP2D6‐specific CD8 T cell reactivity was tested at diagnosis and during treatment and correlated with indices of disease activity. Seven CYP2D6 peptides with high HLA‐A2 binding affinity colocalizing with known B cell or CD4 T cell epitopes were selected. Five sequences inducing high levels of IFN‐γ were used for HLA‐A2 tetramer construction. Frequency, IFN‐γ production, and cytotoxicity of CYP2D6‐specific CD8 T cells were higher at diagnosis than during treatment. Intensity of CYP2D6‐specific CD8 T cell responses correlated with disease activity. Immune responses to CYP2D6245‐254 were the strongest both at diagnosis and during treatment. Conclusion: HLA‐A2–restricted, CYP2D6‐specific CD8 T cell immune responses vary according to disease stage and correlate with hepatocyte damage. CD8 T cell targets on CYP2D6—in particular CYP2D6245‐254—may be the focus of novel immune intervention in AIH‐2. (HEPATOLOGY 2007.)

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 ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor targets.

Cancers are able to grow by subverting immune suppressive pathways, to prevent the malignant cells as being recognized as dangerous or foreign. This mechanism prevents the cancer from being eliminated by the immune system and allows disease to progress from a very early stage to a lethal state. Immunotherapies are newly developing interventions that modify the patients immune system to fight cancer, by either directly stimulating rejection‐type processes or blocking suppressive pathways. Extracellular adenosine generated by the ectonucleotidases CD39 and CD73 is a newly recognized “immune checkpoint mediator” that interferes with anti‐tumor immune responses. In this review, we focus on CD39 and CD73 ectoenzymes and encompass aspects of the biochemistry of these molecules as well as detailing the distribution and function on immune cells. Effects of CD39 and CD73 inhibition in preclinical and clinical studies are discussed. Finally, we provide insights into potential clinical application of adenosinergic and other purinergic‐targeting therapies and forecast how these might develop in combination with other anti‐cancer modalities.

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.