Background: Micro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion. Methods: Mature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRT-PCR and western blotting analysis. Results: A significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells. Conclusions: Decreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.

Although CD8+T cell exhaustion hampers viral control during chronic HBV infection, the pool of CD8+T cells is phenotypically and functionally heterogeneous. Therefore, a specific subpopulation of CD8+T cells should be further investigated. This study aims to dissect a subset of CD8+T cells expressing C-X-C motif chemokine receptor 5 (CXCR5) in chronic HBV infection. The frequency of CXCR5+CD8+T cells and the levels of C-X-C motif chemokine ligand 13 (CXCL13), a chemokine of CXCR5, were measured in patients with chronic HBV infection. C57BL/6, interleukin (IL)-21 receptor- or B cell-deficient mice were hydrodynamically injected with pAAV-HBV1.2 plasmids. Phenotype and functions of peripheral and intrahepatic CXCR5+ and CXCR5-CD8+T cells were assessed. CXCR5+CD8+T cells were partially exhausted but possessed a stronger antiviral ability than the CXCR5- subset in patients with chronic HBV infection; moreover, CXCR5+CD8+T cells were associated with a favorable treatment response in patients with chronic hepatitis B (CHB). High levels of CXCL13 from patients with CHB facilitated the recruitment of intrahepatic CXCR5+CD8+T cells, and this subpopulation produced high levels of HBV-specific interferon (IFN)-γ and IL-21. Notably, PD1 (programmed death 1) blockade and exogenous IL-21 enhanced the production of IFN-γ. More strikingly, mice injected with CXCR5+CD8+T cells showed remarkably decreased expression of HBsAg. Additionally, an impaired production of HBV-specific IFN-γ from intrahepatic CXCR5+CD8+T cells was observed in IL-21 receptor- or B cell-deficient mice. CXCL13 promotes the recruitment of CXCR5+CD8+T cells to the liver, and this subpopulation improves viral control in chronic HBV infection. The identification of this unique subpopulation may contribute to a better understanding of CD8+T cell functions and provide a potential immunotherapeutic target in chronic HBV infection. Exhaustion of CD8+ T cells is an important factor in the development of chronic hepatitis B virus (HBV) infection. CD8+ T cells expressing the receptor CXCR5 are partially exhausted, but have potent antiviral activity, as they produce high levels of HBV-specific cytokines in chronic HBV infection. Increased expression of CXCL13 within the liver facilitates the recruitment of CXCR5+CD8+T cells and establishes effective immune control of HBV infection.

The serological hallmark of primary biliary cirrhosis (PBC) is the presence of high titer and specific antimitochondrial antibodies (AMAs). Although there is no global immune defect in patients with PBC, there is widespread dysregulated B‐cell function, including increased sera levels of immunoglobulin M and enhanced B‐cell responses to cytosine‐phosphate‐guanine stimulation. The mechanisms involved in this B‐cell dysfunction have remained unknown. To address this issue, we focused on identifying the frequencies of B‐cell subsets in patients with PBC and the mechanisms that lead to B‐cell dysregulation, including the relationships with chemokine (C‐X‐C motif) receptor 5 (CXCR5)⁺CD4⁺T cells. Herein, we report that elevations of both serum and intrahepatic interleukin‐21 (IL‐21) were found in patients with PBC and, in particular, promoted B‐cell proliferation, signal transducer and activator of transcription 3 phosphorylation and AMA production in vitro. More important, upon stimulation with recombinant E2 subunit of pyruvate dehydrogenase complex, CXCR5⁺CD4⁺T cells in PBC produced higher levels of IL‐21 than healthy controls. Additionally, sorted CXCR5⁺CD4⁺T cells increased production of AMAs by autologous CD19⁺B cells. Indeed, elevated expression of intrahepatic chemokine (C‐X‐C motif) ligand 13 (CXCL13), a key chemokine of CXCR5⁺ cells, was uniquely found within the portal tracts in PBC, accompanied by infiltrates of CD4⁺, CXCR5⁺, CD19⁺, and CD38⁺ cells. Conclusion: CXCL13 promotes aggregation of CD19⁺B cells and CXCR5⁺CD4⁺T cells, which directs the aberrant AMA response by IL‐21. These data have implications for potential immunotherapy and also reflect the unique lymphoid biology in liver of PBC. (Hepatology 2015;61:1998‐2007)

HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis. Although increasing evidence supports the presence of extracellular HBV RNA (or serum HBV RNA) species and their potentials to be a new marker for monitoring chronic HBV infection, the origin and molecular forms of serum HBV RNA remain ill-defined. In addition to the infectious DNA virions, HBV is known to produce a number of incomplete viral particles extracellularly, including subviral particles (HBsAg), naked capsids, and empty virions, during its replication cycle. Here, we demonstrated that HBV pregonomic (pg) RNA-containing virion-like-particles exist in cell culture fluid and patient sera, which are secreted along with mature DNA virions via the multivesicular body (MVB) secretory route. Moreover, virion-derived HBV RNAs are predominantly spliced and 3’ truncated forms, and HBV RNA virions are unable to initiate a new round of infection in cell cultures. These findings provide new insights into a better understanding of serum HBV RNA biology and a conceptual basis for further widespread clinical applications of serum HBV RNA as a biomarker in chronic hepatitis B patients.

Strategies to improve T cell therapy efficacy in solid tumors such as hepatocellular carcinoma (HCC) are urgently needed. The common cytokine receptor γ chain (γ_c) family cytokines such as IL-2, IL-7, IL-15 and IL-21 play fundamental roles in T cell development, differentiation and effector phases. This study aims to determine the combination effects of IL-21 in T cell therapy against HCC and investigate optimized strategies to utilize the effect of IL-21 signal in T cell therapy. The antitumor function of AFP-specific T cell receptor-engineered T cells (TCR-T) was augmented by exogenous IL-21 in vitro and in vivo. IL-21 enhanced proliferation capacity, promoted memory differentiation, downregulated PD-1 expression and alleviated apoptosis in TCR-T after activation. A novel engineered IL-21 receptor was established, and TCR-T armed with the novel engineered IL-21 receptors (IL-21R-TCR-T) showed upregulated phosphorylated STAT3 expression without exogenous IL-21 ligand. IL-21R-TCR-T showed better proliferation upon activation and superior antitumor function in vitro and in vivo. IL-21R-TCR-T exhibited a less differentiated, exhausted and apoptotic phenotype than conventional TCR-T upon repetitive tumor antigen stimulation. The novel IL-21 receptor in our study programs powerful TCR-T and can avoid side effects induced by IL-21 systemic utilization. The novel IL-21 receptor creates new opportunities for next-generation TCR-T against HCC.