Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro.

CD8(+) T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8(+) T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2(hNTCP) cells that endogenously processed viral antigens and presented them to HBV-specific CD8(+) T cells. This induced cytolytic and noncytolytic CD8(+) T-cell effector functions and reduction of viral loads.

BH3 mimetics reduce adhesion and migration of hepatoblastoma and hepatocellular carcinoma cells.

Advanced stages of tumour and development of metastases are the two major problems in treating liver tumours such as hepatoblastoma (HB) and hepatocellular carcinoma (HCC), in paediatric patients. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We analysed the effect of apoptosis modulators, BH3 mimetics, on mechanisms of dissemination such as adhesion or migration of HB and HCC cells. BH3 mimetics such as ABT-737 and obatoclax can reduce cell migration in a scratch assay as well as adhesion of HB and HCC cells to matrigel. Immunofluorescence staining of F-actin demonstrated that development of lamellipodia, which are important for migration, decreased. BH3 mimetics increase the level of activated caspases 3 and 7 in HUH6 cells. This results in the degradation of GTPase Cdc42, which can be determined by western blot analysis. A pan-caspase inhibitor can block the migration and degradation of Rho-GTPase. In summary, our study showed that BH3 mimetics not only enhance drug sensitivity but also may prevent metastasis by inhibiting HB and HCC cell motility.

The activity of γδ T cells against paediatric liver tumour cells and spheroids in cell culture.

BACKGROUND: Chemoresistance and advanced tumour stage at time of diagnosis are the major reasons for poor treatment results in hepatoblastoma (HB) and paediatric hepatocellular carcinoma (HCC). Positive results with transplantation of liver and bone marrow revealed the impact of the immune system on the treatment of liver malignancies. AIM: Cytotoxic-immune-cells-like natural killer (NK) and T cells are major player in the defence against developing tumours. This study aimed to specifically analyse the ability of ex-vivo expanded γδ T cells to recognise and lyse HB and HCC cell lines in coculture assays. METHODS: Cell viability after treatment with γδ T cells was evaluated with two HB (HUH6 and HepT1) and one HCC cell line (HC-AFW1) using a MTT-based cytotoxicity assay. The binding of T cells to target cells was monitored using immunofluorescence microscopy. RESULTS: Incubation of hepatic tumour cell lines with γδ T cells led to a significant decrease in tumour cell viability. This was enhanced by zoledronic acid and histone deacetylase inhibitors. MT110, an EpCAM/CD3-bispecific BiTE antibody could bluntly enhance tumour cell lysis close to completion. γδ T cells efficiently interacted with HB and HCC cells in a spheroid culture model. CONCLUSION: Bispecific antibodies such as MT110 might be used to intensify the antitumoural effect of γδ T cells in context of adoptive immune cell transfer. Optimised immunotherapeutic strategies might therefore improve the outcome of high risk hepatoblastoma and hepatocellular carcinoma.

Identification of key amino acid residues that determine the ability of high risk HPV16-E7 to dysregulate major histocompatibility complex class I expression.

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.

The role of natural killer cells and CD8(+) T cells in hepatitis B virus infection.

Hepatitis B virus (HBV) infection is one of the main causes of chronic liver diseases that may progress to liver cirrhosis and hepatocellular carcinoma. Host immune responses are important factors that determine whether HBV infection is cleared or persists. Natural killer (NK) cells represent the main effector population of the innate immune system and are abundant in the human liver. Recently, it has been demonstrated that NK cells not only exhibit antiviral functions but may also regulate adaptive immune responses by deletion of HBV-specific CD8(+) T cells. It is well-established that HBV-specific CD8(+) T cells contribute to virus elimination. However, the mechanisms contributing to CD8(+) T cell failure in chronic HBV infection are not well-understood. In this review, we will summarize the current knowledge about NK cells and CD8(+) T cells and illustrate their contribution to viral clearance and persistence in HBV infection. Moreover, novel immunological in vitro model systems and techniques to analyze HBV-specific CD8(+) T cells, which are barely detectable using current multimer staining methods, will be discussed.

Targeting EpCAM (CD326) for immunotherapy in hepatoblastoma.

Hepatoblastoma (HB) is the most common liver cancer in children. Recurrence of HB after chemotherapy and surgery is frequent among high-risk patients and is associated with chemoresistance. Immunotherapy may improve poor treatment outcomes in HB patients. Cytotoxic leukocytes of the innate and adaptive immune system including different populations of cytotoxic T cells play a major role in fighting developing tumors. In this setting, monoclonal antibodies may be employed to specifically direct immune responses toward tumor cells. We addressed this issue by using humanized antibodies that recognize the cell surface molecule EpCAM (CD326, overexpressed in hepatic tumor cells) to enhance immune responses against HB. EpCAM was constantly expressed on HB cells and its expression was independent of previous therapy based on the DNA-damaging agent cisplatin. Co-culture assays performed with two well-described HB cell lines and tumor tissue cultures demonstrated that tumor cell lysis by γδ T cells can be dramatically augmented by applying EpCAM-specific monoclonal antibodies. These data emphasize the value of antitumor immune responses and encourage adapting immunotherapeutic regimens to improve the outcome of high risk HB.