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The EBV-associated Malignancies Program

EBV is associated with several malignant diseases which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the non-immunodominant antigens EBNA-1, LMP-1 and LMP-2 and is seen in EBV-positive Hodgkin’s disease, nasopharyngeal carcinomas and peripheral T/NK-cell lymphomas.

 

1. Immunotherapeutic strategy against EBV latency II malignancies

CD4+ T cells may play a crucial role in controlling EBV latency II malignancies. For this reason, we developed a novel peptidic approach to encourage the induction of an antigen-specific CD4+ T-cell response in these EBV latency II malignancies. Using the prediction software TEPITOPE, we previously selected and combined 6 peptides derived from EBV nuclear antigen-1 (EBNA-1), latency membrane proteins (LMP)-1, and LMP-2, which are highly promiscuous for major histocompatibility complex class II molecules (International extended Patent) and demonstrated their ability to induce interferon-γ–secreting CD4+ T cells (Depil et al. Vaccine 2006; Depil et al. J Immunother 2007).

 

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IFN-γ ELISPOT in adult EBV-positive Hodgkin Lymphoma patients

 

More recently, we have confirmed that all peptides used in this cocktail are recognized by human CD4+ memory T cells from healthy donors, inducing a broad T-helper (Th) 1 response, characterized by interferon-γ and interleukin-2 cytokine secretion. Furthermore, we have generated EBV-specific CD4+ T-cell lines and proved their cytotoxic potential, not only on original models expressing latency II antigens (EBV-transformed T cell or monocyte), but also on lymphoblastoid cell lines expressing latency III antigens. In addition, granzyme B enzyme-linked immunospot assays suggested that a component of this antigen-specific cytotoxic activity could be linked to the granule lytic pathway. Importantly, we have demonstrated that neither phenotypic changes nor functional activities of natural Treg were observed in response to EBV peptides, avoiding any risk of aggravation of the pre-existing immunosuppressive environment reported in EBV-associated malignancies (Moralès et al, J Immunotherapy, 2012).

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Lysis of autologous lymphoblastoid cell lines (LCLs) by EBV-specific CD4+ T cell lines

 

 

2. Implication of Treg in EBV latency II malignancies

A.Treg in Hodgkin’s Lymphoma (HL) linked to EBV infection

CD4+ helper and Treg cells play vital but opposing roles in regulating host immune responses against HL. In 30-40% of patients with HL, EBV is present in the neoplastic cells. However, very little is known about the EBV-induced regulatory mechanisms used in HL to escape immune recognition. Here, we describe the associations of induced regulatory T cells (Tr1) with EBV-positive and EBV-negative HL. In the lymph nodes of EBV-positive HL patients, we found a significant increase of gene expression of Tr1 related markers (CD49b, CD18 and IL-10) compared to the gene expression profile in the lymph nodes dissected from EBV-negative HL subjects. These results were correlated with an increased gene expression of the major immunosuppressive cytokine IL-10. The elevation of IL-10 expression was confirmed by immunohistochemical staining of frozen node biopsies and by flow cytometry analysis of PBMCs from HL patients.

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Intra-nodal expression of Tr1 cell markers determined by immuno-histochemical assay

 

B.Treg in nasopharyngeal carcinoma (NPC)

NPC is an epithelial tumor of the nasopharynx that is associated with EBV infection in nearly 100% of cases. We have confirmed that NPC-derived exosomes have immunoregulatory properties by their ability to induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg chemoattraction. Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.

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Flow Cytometric Analysis of human Treg specific markers in presence of tumor-derived exosomes.

 

 

The HCV-associated Liver Carcinoma Program

Infection HCV is characterized by a high risk of chronicity, which may progress to cirrhosis and development of hepatocellular carcinoma (HCC) in 20 to 25 years. The progression of hepatitis C to chronic status is associated with a profound down-regulation of CD4- and CD8-mediated cellular immunity, limiting anti-HCV cytotoxic capacity. This immunodeficiency may participate in the immune tolerance of HCV and consequently to its persistence. Treg-mediated immunosuppressive mechanisms may be facilitating the immune evasion of HCV, and this hypothesis is under extensive investigation in our team.

 

1. Implication of Treg in hepatitis C liver disease

A.Role of Treg in fibrosis progression to hepatocellular carcinoma (HCC)

We have evaluated the presence of Treg in liver biopsies of three well-defined cohorts of chronically HCV-1b infected patients, including patients without liver lesions, with cirrhosis and with HCC. Using qPCR, we found an increase in the expression of IL-10 and TGF-β in liver biopsies with more severe fibrosis. This observation was correlated with increased expression of specific markers of Treg and Tr1 cells during the pathological progression. Experiments performed on liver biopsies of an 18-year-old HCV-genotype 1b infected patient, followed from the chronic stage until HCC diagnosis, confirmed an increase of Tr1 cells, which was proportionally related to the aggravation of the pathology.

 

 

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Q-PCR Histogram analysis for expression of various immune markers in  liver biopsies of a  genotype 1b-patient at different stages of the pathology

 

 

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Agarose gel electrophoresis analysis showing amplification of cDNA extracted from  infected liver biopsies at different stages of the disease

 

Additionally, we showed for the first time that HCV is able to infect human Treg and promote their recruitment and suppressive activity, contributing to one mechanism by which HCV could escape the immune system, thus leading to worsening of the disease. The contact of VHCcc/JFH-1 (HCV virions produced in cell culture) with human Treg increased the gene expression of HCV receptors, which could lead to an increased susceptibility and increased sensitivity of Treg cells to viral infection. We were able to confirm the internalization of the virus by the identification of viral proteins (E1, E2, Core and NS5A).

 

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HCV – Core protein expression in Treg cells

 

B.Implication of Treg in recurrence of hepatitis C after liver transplantation

Hepatitis C-related cirrhosis is the leading cause for liver transplantation (LT). However, 80% of transplanted patients present with accelerated recurrence of the disease. Using liver biopsies from HCV+ recipients 1 and 5 years after transplantation, we found that Treg markers were overexpressed in all recipients five years after transplantation. On the contrary, Tr1 markers were only overexpressed in subjects with severe recurrence. IL-10 production, a hallmark characteristic of the Tr1 subset, was also enhanced in those with severe recurrence of HCV infection at both 1 and 5 years.

 

 

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TGF-β and interleukin-10 expression in HCV-negative recipients with a mild or severe HCV recurrence after Liver transplantation: (A and B) QPCR- analysis – (C) ELISA assay- (D) Immuno-histochemical staining.

 

In the context of liver transplantation, we have evaluated the effects of cyclosporine A (CsA) and tacrolimus (Tac), the principal immunosuppressive drugs used for organ transplantation, on Treg.  We have observed that low doses of CsA inhibit Treg activity, a finding that might explain the beneficial effect of this drug on hepatitis C recurrence. In contrast, by maintaining Treg activity, Tac could be more helpful than CsA in preventing transplant rejection.

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2. New evaluations of liver disease progression during chronic hepatitis C infection

A.Analysis of gene transcription in sera

Non-invasive techniques are necessary to monitor the progression of liver disease during chronic HCV infection, and serum is the most accessible biological material to study the pathogenesis of HCV. Using qPCR, gene transcription of 219 selected genes involved in the pathogenesis of HCV infection was measured in sera, PBMCs and liver samples collected simultaneously from patients chronically infected with HCV. Analysis of gene transcription revealed comparable, significant correlations between serum and liver, with significant over expression of 41 genes strictly involved in T-cell activation and apoptosis in sera of genotype-1b-infected patients vs. healthy controls. In these patients, alcohol consumption was associated with elevated expression of six genes involved in the inflammatory response, together with abrogated expression of genes associated with dendritic cell function.

B.Prediction of the response to antiviral therapy after liver transplantation

The TRANSPEG study was a prospective study to assess the efficacy of antiviral therapy in patients with recurrent HCV infection after LT. The results indicated that the expression of CD49b, a predominant marker of Tr1, before the introduction of antiviral therapy, is significantly associated with sustained virological response (SVR) at 18 months after treatment initiation. Responders displayed lower serum levels of CD49b than non-responders (findings confirmed in PBMC and liver biopsies). The assessment of CD49b levels is, thus, predictive of the response to antiviral therapy and CD49b may be a biomarker for immune tolerance and for the effectiveness of antiviral therapy during HCV recurrence.

 

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Relative gene expression of the CD49b marker using RT-Q-PCR in Responder and non-responder group of liver transplanted patients

 

C.CD28: A biomarker for predicting “over-immunosuppression” after liver transplantation

At present, no method is available for accurately monitoring the degree of immunosuppression induced by antirejection therapies. Flow cytometry was used to measure the expression of CD28 and CD38 by peripheral blood lymphocytes in 134 stable, long-term survivors of LT. Twenty-two patients developed at least 1 de novo malignancy over a mean interval of 22 ± 14 months after evaluation. The mean frequency of CD28+CD8+cells was significantly lower in the cancer group in comparison to the non-cancer group, but CD38 expression was comparable amongst the two cohorts. Multivariate analyses indicated that an age greater than 50 years and a low frequency of CD28+CD8+ cells at the time of the medical evaluation were the only significant predictors for development of de novo malignancies.

 

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Actuarial representation of the proportion of patients who Developed a de novo malignancy after the check-up point

 

3.Therapeutic strategy in human HCC

H-1 parvovirus, a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents because of its ability to selectively identify and destroy malignant cells.

A.Effect of H-1 PV on human immune system

We have observed that non-activated PBMC are not sensitive to the cytotoxic effect of H-1 PV but that the virus impairs both activated PBMC proliferative ability and viability. Moreover, we revealed that H-1 PV preferentially targets B lymphocytes. Despite these findings, H-1 PV seemed to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly did not affect human neutrophils or one of the major CD4+ T lymphocyte subpopulation. We then have confirmed that H-1 PV activates human CD4+ T cells by increasing activation marker expression and both effective Th1 and Th2 cytokine secretion. In addition, there was no effect of H1-PV on Treg activity. Interestingly, we also have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC. Our results showed for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a helper CD4+ T cell response.

 

 

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Microscopic analysis of human CD4+ T cells  PHA- activated or not and co-cultured with  H1-Parvovirus during 48h

 

B.Validation of oncolytic and oncotropic properties of H-1 PV

We have examined the oncolytic and oncotropic properties of H-1 PV on Hepatocellular carcinoma-derived cell lines and on primary cultures of both tumor explants and healthy liver cells. HCC explants derived from hepatectomy performed in 8 patients were prepared for primary culture and were infected with H-1 PV at various viral loads and observed according to pre-defined kinetics. HCC cell growth was abrogated in comparison with uninfected cells in a dose-dependent manner, depending upon the viral load in the inoculum. In contrast, we did not observe a significant decrease in the viability of healthy hepatocytes, cholangiocytes and intrahepatic fibroblasts after H-1 PV infection.

 

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Test of viability of human hepatocytes isolated from hepato-Cellular Carcinoma resection co-cultured with H1-Parvovirus