Early high levels of regulatory T cells and T helper 1 may predict the progression of recurrent hepatitis C after liver transplantation.

BACKGROUND: Immune response failure against hepatitis C virus (HCV) has been associated with an increased regulatory T cell (Treg) activity. After liver transplantation (LT), 80% of patients experience an accelerated progression of hepatitis C recurrence. The aim of this work was to assess the involvement of Tregs, T helper (Th) 1, 2 and 17 cells in recurrent hepatitis C. METHODS: Peripheral blood cells obtained before and one month after LT from 22 recipients were analysed. Forty-four key molecules related to Treg, Th1, 2 and 17 responses, were evaluated using qRT-PCR. Liver recipients were classified in two groups according to graft fibrosis evaluated by the METAVIR score on the biopsy performed one year after LT (mild: F ≤ 1, n = 13; severe: F > 1, n = 9). Patients developing a severe recurrence were compared with patients with a mild recurrence. RESULTS: mRNA levels of Treg markers obtained one month after LT were significantly increased in patients with a severe disease course when compared to patients with a mild recurrence. Markers of the Th1 response were elevated in the same group. No differences in the markers determined before LT were observed. CONCLUSION: These findings suggest that Treg, induced by a multifactorial process, which could include a strong Th1 response itself, may play a role in suppressing the early antiviral response, leading to a severe recurrence of hepatitis C.

[Specific folic-acid targeted photosensitizer. The first step toward intraperitoneal photodynamic therapy for epithelial ovarian cancer]. / Photosensibilisateur spécifique pour la thérapie photodynamique ciblée des métastases péritonéales des cancers ovariens.

OBJECTIVE: Epithelial ovarian cancer (EOC) management remains association of debulking surgery in combination with platinum-based chemotherapy. Sixty percent of women with EOC considered in remission will develop recurrent disease. An option to improve the completion of cytoreductive surgery may be the use of photodynamic therapy to induce necrosis of peritoneal metastases. A limit of this technique was the toxicity induced by the lack of specificity of old-generation photosensitizer (PS) for tumor tissue if the light could not be specifically applied. To solve this problem, a solution is the design of selective PS. Folate receptor is a promising target for EOC targeted therapy. We present preclinical results concerning properties of a folic-acid targeted photosensitizer. METHOD: Preclinical studies have been performed in vitro on murine and human cell lines of EOC and in vivo with a preclinical model of peritoneal carcinomatosis (Fisher F344 rat/NuTu-19 cell line). They aimed to precise the ability of PS to target specifically tumor tissue, to emit specific fluorescence, and to obtain cell death. RESULTS: Tissue quantification of the PS showed specific incorporation of the folate-targeted PS within tumor tissue. Specificity for ovarian cancer metastases is better than previously reported with others photosensitizers (tumor-to-normal tissue ratio 9.6). We could detect specific fluorescence in vitro and in vivo on peritoneal metastases. Folic-acid targeted PDT allows to obtain human EOC cells death. CONCLUSION: Specific PS may allow the development of efficient and safe intraperitoneal PDT procedure, which could play a role in the prevention of EOC peritoneal recurrences.

Necrosis- and apoptosis-related Met cleavages have divergent functional consequences.

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.

Corticosteroids do not reverse the inhibitory effect of cyclosporine on regulatory T-cell activity in contrast to mycophenolate mofetil.

BACKGROUND: Inevitable hepatitis C virus recurrence after liver transplantation, a major barrier to survival of the transplanted liver may be promoted by immunosuppression and by CD4(+)CD25(+) regulatory T cells (Treg). Treg cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. Moreover, we have previously described low doses of cyclosporine (CsA) to inhibit Treg activity by inducing interleukin-2 and interfron-γ. We investigated here in, the effect of mycophenolate mofetil (MMF) and corticosteroids, usually used in combination with a calcineurin inhibitor on human CD4(+)CD25(+) Treg cells. METHODS: Human CD4(+)CD25(+) cells isolated from healthy donors were cultured in the presence of CsA +/- corticoids or MMF. Suppressive activity of regulatory T cells was assessed in mixed leukocyte reactions including CD25(+) solvents with autologous activated peripheral blood mononuclear cells (PBMC). RESULTS: MMF and dexamethasone inhibited PBMC and Treg proliferation in dose-dependent fashing, maintaining the suppressive activity of Treg cells. However, the association of corticoids with CsA could not reverse the inhibitory effects of CsA on Treg activity, unlike the MMF and CsA combination. CONCLUSION: We have previously shown CsA to significantly impair the function of CD4(+)CD25(+) Treg cells. Herein we reports that corticoids were not able to reverse this effect, whereas MMF couterbalanced it, suggesting that the combination of MMF with CsA maintains regulatory T cells activity promoting tolerance.

Inhibitory effects of cyclosporine on human regulatory T cells in vitro.

BACKGROUND: Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immunosuppression and the emergence of CD4+CD25+ regulatory T cells (Treg). Treg cells can mediate the induction and maintenance of immunological self-tolerance as well as transplant tolerance. We investigated the effects of cyclosporine (CsA), a widely used immunosuppressive agent, on human CD4+CD25+ Treg cells. METHODS: Human CD4+CD25+ cells isolated from healthy donors were cultured in the presence of 40 or 400 ng/mL CsA. The suppressive activity of Treg was assessed in mixed leukocyte reactions (MLR) using CD25+ and autologous activated peripheral blood mononuclear cells (PBMC). Phenotype analysis (flow cytometric, Q-PCR) and cytokine production (ELISA) of Treg cells were then performed on cultures. RESULTS: CsA (40 or 400 ng/mL) inhibited the proliferative capacity of PBMC and CD4+CD25+ Treg in a dose-dependent manner. Interestingly, addition of 40 ng/mL CsA in MLR impaired the suppressive activity of CD4+CD25+ cells, whereas a higher dose of CsA had no effect on Treg function. It appears that a therapeutic dose of CsA (40 ng/mL) did not change the phenotype of CD4+CD25+ T cells, but altered Treg activity by switching the regulatory to an inflammatory cytokine profile. CONCLUSION: CsA significantly impaired the function of CD4+CD25+ Treg cells by inducing interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence.

Increased expression of regulatory Tr1 cells in recurrent hepatitis C after liver transplantation.

Immune response failure during HCV infection has been associated with the activity of regulatory T cells. Hepatitis C-related cirrhosis is the main reason for liver transplantation. However, 80% of transplanted patients present an accelerated recurrence of the disease. This study assessed the involvement of regulatory T-cell subsets (CD4+CD25+ cells: 'Treg' and CD49b+CD18+ cells: 'T regulatory-1' cells), in the recurrence of HCV after liver transplantation, using transcriptomic analysis, ELISA assays on serum samples and immunohistochemistry on liver biopsies from liver recipients 1 and 5 years after transplantation. Three groups of patients were included: stable HCV-negative recipients and those with mild and severe hepatitis C recurrence. At 5 years, Treg markers were overexpressed in all HCV+ recipients. By contrast, Tr1 markers were only overexpressed in patients with severe recurrence. At 1 year, a trend toward the overexpression of Tr1 was noted in patients evolving toward severe recurrence. IL-10 production, a characteristic of the Tr1 subset, was enhanced in severe recurrence at both 1 and 5 years. These results suggest that Tr1 are enhanced during severe HCV recurrence after liver transplantation and could be predictive of HCV recurrence. High levels of IL-10 at 1 year could be predictive of severe recurrence, and high IL-10 producers might warrant more intensive management.

[Regulatory T-cells and hepatocellular carcinoma: implication of the regulatory T lymphocytes in the control of the immune response]. / T régulateurs et carcinome hépatocellulaire : rôle des lymphocytes T régulateurs dans le contrôle de la réponse immune.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and also the third most common cause of cancer-related death. HCC arises most frequently in males with cirrhosis, which is most often a consequence of chronic hepatitis infection (HBV and HCV) or alcohol abuse. To date, the only effective approaches for patients with HCC are resection or liver transplantation. Immunological mechanisms are important in the surveillance of malignancy and control of tumor progression. Tumor-infiltrating lymphocytes (TILs) have been described in HCC, and extensive infiltration has been associated with reduced tumor recurrence following resection. However continued tumor-growth, despite the presence of a lymphocytic infiltration, including tumor-specific T-cells within and surrounding tumors, suggests a failure of immune control. Although, many mechanisms have been proposed for this attenuated immune response, it becomes evident that direct suppression of effector cells, supported by regulatory T-cells could play a pivotal role in the suppression of immune response to tumors. Initially described in context of immune disorders such as inflammatory autoimmune pathologies, regulatory T lymphocytes are characterized by their capacity to inhibit T helper response. To date, several regulatory T-cells are described, however CD4+CD25+ regulatory T-cells and Tr1 subpopulations remain best characterized. Currently, there is no evidence for direct implication of CD4+CD25+ regulatory T-cells in the malignancy and control of HCC progression. However, recent studies showed that both regulatory T-cells subpopulations and particularly Tr1 have been implicated in the modulation of the immune response during HCV chronic infection, supporting HCC progression.

Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major etiological agent of chronic liver disease and hepatocellular carcinoma (HCC). We demonstrate herewith that HCV core proteins encoded by sequences isolated from HCC tumor tissues, but not those derived from their non-tumor counterparts in the same liver, co-localise in vitro and in vivo and co-immunoprecipitate with PKR in hepatocytic Huh7 cells. We show that this association in fact augments the autophosphorylation of PKR and the phosphorylation of the translation initiation factor eIF2alpha, which are two markers of PKR activity. The present study therefore identifies a novel model of virus-cell interactions whereby a viral protein, the HCV core, activates PKR activity.

Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner.

The impact of hepatitis C virus NS5A protein mutations on interferon alfa (IFN-alpha) signaling pathway, cell proliferation, and viability is an important issue that is still under debate. We have therefore combined transient and stable expression in a human hepatocytic cell line (Huh7) of 3 full-length NS5A sequences, isolated from patients with or without response to IFN-alpha therapy. Expression of all 3 NS5A-reduced IFN-alpha global antiviral activity on both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) replication. We did not show, however, an effect of these 3 NS5A proteins on double-stranded RNA-dependent kinase (PKR) expression and activity as well as colocalization and coimmunoprecipitation between NS5A and PKR. We also failed to show an effect of the 3 NS5A mutants tested on cell proliferation and viability. Overall, our results support an important role of NS5A in controlling IFN-alpha antiviral activity; they show, however, that PKR-independent mechanisms are implicated, at least in liver-derived cells.

Dermal endothelial cells and keratinocytes produce IL-7 in vivo after human Schistosoma mansoni percutaneous infection.

The parasite Schistosoma mansoni infects its definitive mammalian host through an obligatory cutaneous penetration. In this work, we studied early immune response following migration of larvae through human skin, the first immunocompetent organ encountered by the parasite. For this purpose we used an experimental model of severe combined immunodeficient mice engrafted with human skin and injected with autologous PBL. Six days after percutaneous infection, we observed an infiltration of lymphocytes within the human skin, predominantly composed of CD4+ T cells. Moreover, among the cytokines potentially present in the infected skin, immunohistochemistry analysis revealed an in vivo expression of IL-7 in the epidermal layers and strikingly at the level of vascular endothelium. Using an in vitro coculture system, we showed that the S. mansoni larvae directly trigger IL-7 production by human dermal microvascular endothelial cells but not by keratinocytes. Finally, measurements of IL-7 concentrations in plasma of 187 S. mansoni-infected individuals showed that the youngest, which are also the most infected, displayed the highest IL-7 levels. Together, these findings describe dermal endothelial cells as a novel source of IL-7, a cytokine particularly important in schistosomiasis.

Primary Th1 cell immunization against HIVgp160 in SCID-hu mice coengrafted with peripheral blood lymphocytes and skin.

SCID-hu mouse models are of interest in the pathologic investigation of HIV infection, but obtaining a T cell response in SCID-hu-PBL mice is still controversial. We have developed a SCID model by engrafting human skin and autologous PBLs from HIV-seronegative individuals. The study describes the ability of this human-mouse chimera to generate in vivo a primary T lymphocyte response against HIV Ag. The injection of human autologous PBLs was performed 4 to 5 wk after the skin engraftment. Two weeks after injection of PBLs, chimeric mice were immunized with recombinant canary pox virus expressing HIV-1 LAIgp160 (vCP-LAIgp160) and supplemented or not with rIL-2. Intradermal vCP-LAIgp160 injection induced an intradermal perivascular human lymphocytic infiltrate and an epidermic network of CD1a+, CD80+, and CD86+ cells. We derived CD4+ T cell lines (STLs) from the human skin graft of immunized mice, showing that STLs mediated an MHC class II-restricted cytolytic activity directed against HIV-LAIgp160 Ags. Cytokine gene expression in both human skin cells and in STLs showed a predominance of IL-2, IFN-gamma, and IL-12 transcripts. Finally, the T cell repertoire analysis using the immunoscope technique showed a very limited CDR3 length polymorphism in the skin infiltrating lymphocytes suggesting an Ag-specific repertoire. The ability to induce a primary Th1 cell response in vivo affords a useful preclinical model for testing vaccine strategies.