Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.

Preribosomes escaping from the nucleus are caught during translation by cytoplasmic quality control.

Assembly of fully functional ribosomes is a prerequisite for failsafe translation. This explains why maturing preribosomal subunits have to pass through an array of quality-control checkpoints, including nuclear export, to ensure that only properly assembled ribosomes engage in translation. Despite these safeguards, we found that nuclear pre-60S particles unable to remove a transient structure composed of ITS2 pre-rRNA and associated assembly factors, termed the 'foot', escape to the cytoplasm, where they can join with mature 40S subunits to catalyze protein synthesis. However, cells harboring these abnormal ribosomes show translation defects indicated by the formation of 80S ribosomes poised with pre-60S subunits carrying tRNAs in trapped hybrid states. To overcome this translational stress, the cytoplasmic surveillance machineries RQC and Ski-exosome target these malfunctioning ribosomes. Thus, pre-60S subunits that escape nuclear quality control can enter translation, but are caught by cytoplasmic surveillance mechanisms.

Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit.

Nuclear export of preribosomal subunits is a key step during eukaryotic ribosome formation. To efficiently pass through the FG-repeat meshwork of the nuclear pore complex, the large pre-60S subunit requires several export factors. Here we describe the mechanism of recruitment of the Saccharomyces cerevisiae RNA-export receptor Mex67-Mtr2 to the pre-60S subunit at the proper time. Mex67-Mtr2 binds at the premature ribosomal-stalk region, which later during translation serves as a binding platform for translational GTPases on the mature ribosome. The assembly factor Mrt4, a structural homolog of cytoplasmic-stalk protein P0, masks this site, thus preventing untimely recruitment of Mex67-Mtr2 to nuclear pre-60S particles. Subsequently, Yvh1 triggers Mrt4 release in the nucleus, thereby creating a narrow time window for Mex67-Mtr2 association at this site and facilitating nuclear export of the large subunit. Thus, a spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit.

Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function.

A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.

Heat shock factor gene family in rice: genomic organization and transcript expression profiling in response to high temperature, low temperature and oxidative stresses.

Binding of heat shock factors (HSFs) with heat shock element sequence is critical for the transcriptional induction of heat shock genes. Rice genome sequence shows 26 OsHsf genes out of which 25 possess various important domains noted in HSFs i.e. DNA binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), nuclear export signal (NES) and AHA type activation domain. OsHsf entry LOC_Os06g226100 has the oligomerization domain but lacks the above other domains. Also, there are no ESTs or full-length cDNA noted for this entry in database. Expression profiling showed that 22 OsHsf genes are induced by high temperature. Induction of 10 and 14 OsHsf genes was also noted against low temperature stress and oxidative stress, respectively. All OsHsf genes induced by oxidative stress were also induced by high temperature. On the other hand, induction of 6 and 1 OsHsf genes was noted to be exclusive to high and low temperature stresses, respectively. Seven OsHsf genes showed induced expression in response to all the three stresses examined. While in silico promoter analysis showed that OsHsf genes contain upstream regulatory elements corresponding to different abiotic stresses, there was lack of correlation noted between the in silico profiling of the elements and their corresponding transcript expression patterns. Apart from stress inducibility, EST database suggests that various OsHsf genes are developmentally regulated in diverse tissue types.