Nonlytic Lymphocytes Engineered to Express Virus-Specific T-Cell Receptors Limit HBV Infection by Activating APOBEC3.

BACKGROUND & AIMS: Strategies to develop virus-specific T cells against hepatic viral infections have been hindered by safety concerns. We engineered nonlytic human T cells to suppress replication of hepatitis B virus (HBV) and hepatitis C virus (HCV) without overt hepatotoxicity and investigated their antiviral activity. METHODS: We electroporated resting T cells or T cells activated by anti-CD3 with mRNAs encoding HBV or HCV-specific T-cell receptors (TCRs) to create 2 populations of TCR-reprogrammed T cells. We tested their ability to suppress HBV or HCV replication without lysis in 2-dimensional and 3-dimensional cultures of HepG2.2.15 cells and HBV-infected HepG2-hNTCP cells. We also injected TCR-reprogrammed resting and activated T cells into HBV-infected urokinase-type plasminogen activator/severe combined immunodeficiency disease/interleukin 2γ mice with humanized livers and measured levels of intrahepatic and serological viral parameters and serum alanine aminotransferase. Livers were collected for analysis of gene expression patterns to determine effects of the TCR-reprogrammed T cells. RESULTS: TCR-reprogrammed resting T cells produced comparable levels of interferon gamma but lower levels of perforin and granzyme than activated T cells and did not lyse HCV- or HBV-infected hepatoma cells. Although T-cell secretion of interferon gamma was required to inhibit HCV replication, the HBV-specific TCR-reprogrammed resting T cells reduced HBV replication also through intracellular activation of apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3). The mechanism of APOBEC3 intracellular activation involved temporal expression of lymphotoxin-ß receptor ligands on resting T cells after TCR-mediated antigen recognition and activation of lymphotoxin-ß receptor in infected cells. CONCLUSIONS: We developed TCR-reprogrammed nonlytic T cells capable of activating APOBEC3 in hepatoma cells and in HBV-infected human hepatocytes in mice, limiting viral infection. These cells with limited hepatotoxicity might be developed for treatment of chronic HBV infection.

Intrahepatic CD206+ macrophages contribute to inflammation in advanced viral-related liver disease.

BACKGROUND & AIMS: Liver inflammation is key in the progression of chronic viral hepatitis to cirrhosis and hepatocellular carcinoma. The magnitude of viral replication and the specific anti-viral immune responses should govern the degree of inflammation, but a direct correlation is not consistently found in chronic viral hepatitis patients. We aim to better define the mechanisms that contribute to chronic liver inflammation. METHODS: Intrahepatic CD14+ myeloid cells from healthy donors (n=19) and patients with viral-related liver cirrhosis (HBV, HBV/HDV or HCV; n=15) were subjected to detailed phenotypic, molecular and functional characterisation. RESULTS: Unsupervised analysis of multi-parametric data showed that liver disease was associated with the intrahepatic expansion of activated myeloid cells mainly composed of pro-inflammatory CD14+HLA-DRhiCD206+ cells, which spontaneously produced TNFα and GM-CSF. These cells only showed heightened pro-inflammatory responses to bacterial TLR agonists and were more refractory to endotoxin-induced tolerance. A liver-specific enrichment of CD14+HLA-DRhiCD206+ cells was also detected in a humanised mouse model of liver inflammation. This accumulation was abrogated following oral antibiotic treatment, suggesting a direct involvement of translocated gut-derived microbial products in liver injury. CONCLUSIONS: Viral-related chronic liver inflammation is driven by the interplay between non-endotoxin-tolerant pro-inflammatory CD14+HLA-DRhiCD206+ myeloid cells and translocated bacterial products. Deciphering this mechanism paves the way for the development of therapeutic strategies specifically targeting CD206+ myeloid cells in viral-related liver disease patients. Lay summary: Viral-related chronic liver disease is driven by intrahepatic pro-inflammatory myeloid cells accumulating in a gut-derived bacterial product-dependent manner. Our findings support the use of oral antibiotics to ameliorate liver inflammation in these patients.

Multispot, label-free biodetection at a phantom plastic-water interface.

Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.

G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma.

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we find that short-term inhibition of G9a/GLP increases T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increases granzyme expression without terminal T cell differentiation or exhaustion and results in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

Splice-Switching Antisense Oligonucleotides as a Targeted Intrinsic Engineering Tool for Generating Armored Redirected T Cells.

Modification of specificity of T cells for the use in adoptive transfer (CAR- or TCR-redirected T cells) has revolutionized the therapy of liquid tumors and some infectious diseases. However, several obstacles are still hampering the efficacy of such potent therapy, hence concurrent modification of the function is also required to obtain successful results. Here we show the use of splice-switching antisense oligonucleotides (SSOs) as a tool to transiently modify T cell function. We demonstrate the possibility to transfect SSOs and an exogenous TCR into primary human T cells in the same electroporation reaction, without affecting viability and function of the transfected T lymphocytes. Moreover, we show that SSOs targeting T cell-specific mRNAs induce the skipping of the targeted exons, and the reduction of the protein and consequent modification of T cell function. This technical work paves the way to the use of SSOs in immune cells, not only for the knockdown of the functional isoform of the targeted proteins, but also for the protein manipulation by elimination of specific domains encoded by targeted exons.

CD4+ T Cells Mediate the Development of Liver Fibrosis in High Fat Diet-Induced NAFLD in Humanized Mice.

Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4+ T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4+ central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ). In vivo depletion of human CD4+ T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4+ memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics.

Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model.

In the hepatitis B virus (HBV)-related hepatocellular carcinoma tumor microenvironment (TME), monocytes reportedly impede natural T cell functions via PD-L1/PD-1 signaling. However, it remains unclear if T cell receptor-redirected T cells (TCR T cells) are similarly inhibited. Hence, we developed a 3D intrahepatic TME microfluidic model to investigate the immunosuppressive potential of monocytes toward HBV-specific TCR T cells and the role of PD-L1/PD-1 signaling. Interestingly, in our 3D static microfluidic model, we observed that monocytes suppressed only retrovirally transduced (Tdx) TCR T cell cytotoxicity toward cancer cells via PD-L1/PD-1, while mRNA electroporated (EP) TCR T cell cytotoxicity was not affected by the presence of monocytes. Importantly, when co-cultured in 2D, both Tdx and EP TCR T cell cytotoxicity toward cancer cells were not suppressed by monocytes, suggesting our 3D model as a superior tool compared to standard 2D assays for predicting TCR T cell efficacy in a preclinical setting, which can thus be used to improve current immunotherapy strategies.

Corrigendum: Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model.

[This corrects the article DOI: 10.3389/fimmu.2018.00416.].

Lymphocytes transiently expressing virus-specific T cell receptors reduce hepatitis B virus infection.

Adoptive transfer of T cells engineered to express a hepatitis B virus-specific (HBV-specific) T cell receptor (TCR) may supplement HBV-specific immune responses in chronic HBV patients and facilitate HBV control. However, the risk of triggering unrestrained proliferation of permanently engineered T cells raises safety concerns that have hampered testing of this approach in patients. The aim of the present study was to generate T cells that transiently express HBV-specific TCRs using mRNA electroporation and to assess their antiviral and pathogenetic activity in vitro and in HBV-infected human liver chimeric mice. We assessed virological and gene-expression changes using quantitative reverse-transcriptase PCR (qRT-PCR), immunofluorescence, and Luminex technology. HBV-specific T cells lysed HBV-producing hepatoma cells in vitro. In vivo, 3 injections of HBV-specific T cells caused progressive viremia reduction within 12 days of treatment in animals reconstituted with haplotype-matched hepatocytes, whereas viremia remained stable in mice receiving irrelevant T cells redirected toward hepatitis C virus-specific TCRs. Notably, increases in alanine aminotransferase levels, apoptotic markers, and human inflammatory cytokines returned to pretreatment levels within 9 days after the last injection. T cell transfer did not trigger inflammation in uninfected mice. These data support the feasibility of using mRNA electroporation to engineer HBV TCR-redirected T cells in patients with chronic HBV infection.

A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors.

The tumor microenvironment imposes physical and functional constraints on the antitumor efficacy of adoptive T cell immunotherapy. Preclinical testing of different T cell preparations can help in the selection of efficient immune therapies, but in vivo models are expensive and cumbersome to develop, while classical in vitro 2D models cannot recapitulate the spatiotemporal dynamics experienced by T cells targeting cancer. Here, we describe an easily customizable 3D model, in which the tumor microenvironment conditions are modulated and the functionality of different T cell preparations is tested. We incorporate human cancer hepatocytes as a single cell or as tumor cell aggregates in a 3D collagen gel region of a microfluidic device. Human T cells engineered to express tumor-specific T cell receptors (TCR-T cells) are then added in adjacent channels. The TCR-T cells' ability to migrate and kill the tumor target and the profile of soluble factors were investigated under conditions of varying oxygen levels and in the presence of inflammatory cytokines. We show that only the 3D model detects the effect that oxygen levels and the inflammatory environment impose on engineered TCR-T cell function, and we also used the 3D microdevice to analyze the TCR-T cell efficacy in an immunosuppressive scenario. Hence, we show that our microdevice platform enables us to decipher the factors that can alter T cell function in 3D and can serve as a preclinical assay to tailor the most efficient immunotherapy configuration for a specific therapeutic goal.

Multi-spot, label-free immunoassay on reflectionless glass.

Biosensing platforms that combine high sensitivity, operational simplicity and affordable costs find wide application in many fields, including human diagnostics, food and environmental monitoring. In this work, we introduce a label-free biosensing chip made of glass with a single anti-reflective layer of SiO2. This common and economic material coated by a multi-functional copolymer based on dimethylacrylamide enables the detection even in turbid media. The copolymer coating provides covalent immobilization of antibodies onto the surface and prevents the non-specific adsorption of analytes and matrix constituents. The specific capture of target compounds yields a local increase of surface reflectivity measured by a simple imaging system. Chip design and quantitative interpretation of the data are based on a theoretical optical model. This approach enables the multiplex detection of biomolecular interactions with state-of-the-art sensitivity and minimal instrumental complexity. The detection performance is demonstrated by characterizing the interaction between human growth hormone in solution and the corresponding antibodies immobilized on the sensing surface, both in buffer and human serum, obtaining a clear signal for concentrations as small as 2.8 ng/ml.

A fast and simple label-free immunoassay based on a smartphone.

Despite the continuous advancements in bio-molecular detection and fluidic systems integration, the realization of portable and high performance devices for diagnostic applications still presents major difficulties, mostly because of the need to combine adequate sensitivity with low cost of production and operational simplicity and speed. In this context, we propose a compact device composed of a smartphone and a custom-designed cradle, containing only a disposable sensing cartridge, a tiny magnetic stirrer and a few passive optical components. The detection principle is the previously proposed Reflective Phantom Interface that is based on measuring the intensity of light reflected by the surface of an amorphous fluoropolymer substrate, which has a refractive index very close to that of the aqueous sample solution and hosts various antibodies immobilized within spots. The reflectivity of dozens of spots is monitored in real time by the phone׳s camera using the embedded flash LED as the illumination source. We test the performance of the combined device targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV. Target concentrations as low as a few ng/ml can be rapidly and robustly determined by comparing the rate of increase of the signal after the addition of the sample with that measured after the subsequent addition of a standard solution with known concentration. The features of the proposed system enable the realization of novel handheld biosensing devices suitable for those applications where multiple targets have to be rapidly detected even without the presence of trained personnel.