Search

The maturation process of natural killer (NK)cells determines their functionality,during which multiple transcriptional factors play a critical role. However, few checkpoints specifically targeting this process have been discovered. Here, we show that NK-specific deficiency of glucose-regulated protein 94 (gp96) led to decreased maturation of NK cells in mice. These gp96-deficient NK cells exhibited undermined activation, cytotoxicity, and IFN-γ production upon stimulation and weakened response to IL-15 for maturation. NK-specific gp96-deficient mice were prone to tumor growth in vivo. Eomes was identified as a key transcription factor involved in gp96-mediated NK maturation. Interaction between gp96 and E3 ubiquitin ligase Trim28 blocked Trim28 binding to Eomes and protected Eomes from ubiquitination and degradation. Together, our study demonstrates that the gp96-Trim28-Eomes axis plays a critical role in NK cell maturation and anti-tumor immunity in mice, suggesting a novel mechanism for gp96 in regulating NK cell immunity.

Breast cancer is the most common tumor in female, which seriously threatens the health of women. Triple-negative breast cancer is a subtype with the worst prognosis because of its special physiological characteristics and lack of targeted drugs. Therefore, it is urgent to develop new targeted treatments to improve the prognosis and survival rate of the patients. Previous studies have shown that heat shock protein gp96 is expressed on the membrane of a variety of cancer cells but not on the normal cells. Cell membrane gp96 levels are closely related to the poor prognosis of breast cancer, which may serve as a new target for breast cancer treatment. Based on the structure of gp96, we designed an α-helical peptide p37 that specifically targeting the ATP binding region of gp96. To improve the stability and decrease the degradation of the peptide, the N-terminus or C-terminus of p37 was coupled to PEG

AFP is the most widely used biomarker for the diagnosis of hepatocellular carcinoma. However, a substantial proportion of HCC patients have either normal or marginally increased AFP levels in serum, and the underlying mechanisms are not fully understood. In the present study, we provided in vitro as well as in vivo evidence that heat shock protein gp96 promoted AFP expression at the transcriptional level in HCC. NR5A2 was identified as a key transcription factor regulated by AFP and its stability was enhanced by gp96. A further mechanistic study by CO-IP, GST-pull down and molecular docking showed the competitive binding of gp96 and SUMO E3 ligase RanBP2 to NR5A2 at the sites spanning from aa 507 to 539. The binding of gp96 inhibited SUMOylating, ubiquitination, and subsequent degradation of NR5A2. In addition, clinical analysis of HCC patients indicated that gp96 expression was positively correlated to serum AFP levels in tumors. Therefore, our study uncovered the novel regulatory mechanism of gp96 on the stability of its client proteins by directly affecting their SUMOylation and ubiquitination. These findings will help in designing more accurate AFP-based HCC diagnosis and progression monitoring approaches.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from cold-chain foods to frontline workers poses a serious public health threat during the current global pandemic. There is an urgent need to design concise approaches for effective virus inactivation under different physicochemical conditions to reduce the risk of contagion through viral contaminated surfaces of cold-chain foods. By employing a time course of electron beam exposure to a high titer of SARS-CoV-2 at cold-chain temperatures, a radiation dose of 2 ​kGy was demonstrated to reduce the viral titer from 10

Summary Upregulation and stabilization of Foxp3 expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, gp96 immunization showed obvious therapeutic effects in a Lyn–/– mouse model of systemic lupus erythematosus. Moreover, gp96 alleviated the initiation and progression of MOG-induced experimental autoimmune encephalomyelitis. Immunization of gp96 increased Treg frequency, expansion, and suppressive function. Gene expression profiling identified the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody-induced and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases., Graphical abstract, Highlights • SLE symptoms in Lyn–/– mice are ameliorated by gp96 immunization • Tregs expanded by gp96 provide potential in suppressing Th-mediated EAE • Gp96 promotes Treg proliferation, stability, and suppressive function • Gp96 binds to and activates Treg in a TLR2-MyD88-NF-кB-Foxp3 pathway, Immune response, Genomics, Molecular biology

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8(+) T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8(+) T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8(+) T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8(+) T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection. IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths and the fifth most common cancer worldwide. Clinically therapeutic options for HCC are very limited, and the overall survival rate of patients is very low. Therefore, early diagnosis and treatment of HCC have important impact on overall survival of patients. At present, alpha-fetoprotein (AFP) is one of the most widely used serological markers for HCC. Many evidences have shown that as a specific onco-protein, AFP has great research value in the occurrence, development, diagnosis and treatment of HCC. Here, we briefly introduce the molecular mechanism of AFP in the regulation of HCC occurrence and development, and its role in tumor escape from immune surveillance. We focus on the application of AFP as an important HCC target or carcino-embryonic antigen (CEA) in HCC clinical diagnosis and treatment.

In hepatitis B virus (HBV) infection, the virus produces redundant hepatitis B surface antigen (HBsAg) that plays a key role in driving T-cell tolerance and viral persistence. However, currently available anti-HBV agents have no direct effect on HBsAg transcription and protein expression. In this study, we designed a heat shock protein gp96 inhibitor p37 with the cell penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p37 internalization into hepatocytes. PTD-p37 effectively suppressed HBsAg expression and viral replication both in vitro and in vivo. We further provide evidence that PTD-p37 suppressed HBV enhancer/promoter activity via p53 upregulation. Moreover, PTD-p37 had antiviral activity against a lamivudine-resistant HBV strain. Considering that suppression of HBsAg expression is a major goal for treatment of HBV infection, our results provide a basis for developing a new therapeutic approaches targeting host factors against viral expression.

Kupffer cells (KCs), the largest tissue-resident macrophage population in the body, play a central role in maintaining a delicate balance between immune tolerance and immunity in the liver. However, the underlying molecular mechanism remains elusive. In this study, we show that KCs express high levels of miR-146a, which is under control of the PU.1 transcription factor. miR-146a deficiency promoted KCs differentiation toward a proinflammatory phenotype; conversely, miR-146a overexpression suppressed this phenotypic differentiation. We found that hepatitis B virus (HBV) persistence or HBV surface Ag treatment significantly upregulated miR-146a expression and thereby impaired polarization of KCs toward a proinflammatory phenotype. Furthermore, in an HBV carrier mouse model, KCs depletion by clodronate liposomes dramatically promoted HBV clearance and enhanced an HBV-specific hepatic CD8+ T cell and CD4+ T cell response. Consistent with this finding, miR-146a knockout mice cleared HBV faster and elicited a stronger adaptive antiviral immunity than wild-type mice. In vivo IL-12 blockade promoted HBV persistence and tempered the HBV-specific CTL response in the liver of miR-146a knockout mice. Taken together, our results identified miR-146a as a critical intrinsic regulator of an immunosuppressive phenotype in KCs under inflammatory stimuli, which may be beneficial in maintenance of liver homeostasis under physiological condition. Meanwhile, during HBV infection, miR-146a contributed to viral persistence by inhibiting KCs proinflammatory polarization, highlighting its potential as a therapeutic target in HBV infection.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection.IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8

Utilizing regulatory T cells (Treg) is a promising approach for treating autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatic diseases. Upregulation and stabilization of forkhead box P3 (Foxp3) expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, we examined the potential effect and mechanism of heat shock protein gp96 in regulating Foxp3 expression and Treg activation in vivo. High-dose gp96 immunization showed obvious therapeutic effects in a Lyn-/- mouse model of SLE, as evidenced by decreased auto-antibody titers, follicular helper T cells, and antibody-production plasma cells. Moreover, immunization of mice with gp96 alleviated the initiation and progression of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE). Immunization of gp96 increased Treg frequency, expansion, and suppressive function with no obvious effector T cell activation. Gene expression profiling was used to identify the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout (KO) mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody- and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases.

Toll like receptors (TLRs) are pattern recognition receptors and represent immune receptors in innate immunity. They are very conservative in evolution and extremely important for the survival of organisms. TLRs initiate signal transduction through binding of endogenous or exogenous ligands to activate a series of downstream important gene expression and activation. Studies have shown that regulatory T cells (Tregs) play a central role in maintaining peripheral immune tolerance and preventing transplant rejection. Tregs express certain TLRs, including TLR2, TLR4, TLR5, TLR7, TLR8, and TLR9. Activation of TLRs may directly or indirectly affect (mainly activate) Treg proliferation and immunosuppressive functions, and this regulation is closely related to the occurrence of infection, autoimmune disease and cancer. The heat shock proteins as TLRs ligand molecules play important roles in the regulation of Treg. Therefore, understanding regulatory mechanisms of TLR pathways on Tregs is of great significance for new drug development and targeted therapy. This review introduces how TLR-mediated pathways regulate Tregs' immune function.模式识别受体Toll 样受体 (Toll like receptors，TLRs) 是固有免疫中免疫受体的代表，进化上十分保守，对生物体的生存极为重要。TLRs 通过内源或外源的配体启动信号转导，激活下游一系列重要的基因表达与活化。研究表明调节性T 细胞 (Regulatory T cell，Treg) 在维持机体外周免疫耐受和阻止移植排斥反应等方面发挥核心作用。Treg 细胞表达某些TLRs，包括TLR2、TLR4、TLR5、TLR7、TLR8、TLR9 等。TLRs 的活化可能直接或间接地影响 (主要是活化) Treg 的增殖和免疫抑制功能，这种调节与感染、自身免疫病和癌症的发生密切相关。其中热休克蛋白作为TLRs 配体分子对于Treg 的调节发挥了重要的作用。因此，了解TLRs 通路对研究Treg 免疫调控机制、新药物研发和靶向治疗有重大意义。文中简要介绍了TLRs 通路调节Treg 免疫功能的相关研究进展。.

Liver failure leads to the massive necrosis of hepatocytes, releasing large amounts of intracellular components including damage-associated molecular patterns (DAMPs). We found that extracellular gp96 levels in serum were elevated in patients with chronic hepatitis B infection (CHB) and acute-on-chronic liver failure (ACLF). Meanwhile, the gp96 level positively correlated with hepatic necroinflammation. We employed two mouse liver damage and liver failure models induced by lipopolysaccharide (LPS) plus d-galactosamine (d-Galn), and concanavalin A (ConA) to identify the function of extracellular gp96. As a result, the inhibition of extracellular gp96 by a specific peptide efficiently mitigated both LPS/d-Galn- and ConA-induced liver injury and immune hyperactivation, whereas exogenous gp96 aggravated the symptoms of hepatic injury in mice but not in Kupffer cells-ablated mice. The exposure of Kupffer cells to gp96 induced the secretion of pro-inflammatory cytokines. Collectively, our data demonstrate that gp96 released from necrotic hepatocytes aggravates immune hyperactivation and promotes liver damage and possibly the development of liver failure mainly by activating Kupffer cells.

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus (HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.

// Xin Li 1, * , Bo Wu 1, * , Lizhao Chen 1 , Ying Ju 1 , Changfei Li 1 and Songdong Meng 1, 2 1 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China 2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China * These authors have contributed equally to this work Correspondence to: Songdong Meng, email: mengsd@im.ac.cn Changfei Li, email: lichangfei2006@163.com Keywords: uPAR,miR-17-5p, miR-20a, DR5, apoptosis Received: December 21, 2016 Accepted: July 23, 2017 Published: August 24, 2017 ABSTRACT Dissection and understanding of the molecular pathways driving triple-negative breast cancer (TNBC) are urgently needed to develop efficient tailored therapies. Aside from cell invasion and metastasis, the urokinase-type plasminogen activator receptor (uPAR) has been linked to apoptosis resistance in breast tumors. We explored the mechanism of uPAR-disrupted apoptosis in breast cancer. We found that depletion of uPAR by RNAi increases death receptor 4 (DR4) and death receptor 5 (DR5) expression and triggers TRAIL-induced apoptosis in TNBC cells. The microRNAs miR-17-5p and miR-20a inhibit cell apoptosis via suppression DR4/DR5. We provide evidence that uPAR enhances miR-17-5p/20a expression through upregulation of c-myc. Blocking miR-17-5p/20a with antagomiRNA suppressed the growth of uPAR-overexpressing breast tumor xenografts in mice. These results indicate that uPAR suppresses cell apoptosis by inhibiting the c-myc-miR-17/5p/20a-DR4/DR5 pathway. Therapy directed at uPAR-induced miR-17/20a is a potential option for breast cancer and TNBC.

Bispecific antibody (BsAbs) are antibodies (Abs) containing two different antigen-binding sites in one molecule. In the last decade, three BsAbs drugs have been approved for therapeutic use. Meanwhile there are a number of BsAbs in preclinical or clinical studies. In this review, we describe BsAb design, discovery, mechanism of action, and the recent research progress in developing BsAbs.双特异抗体是指可以同时结合两个不同抗原或一个抗原不同表位的特殊抗体，目前已有3 个双特异抗体批准上市，还有很多个双特异抗体处于临床或临床前研究阶段。文中就双特异抗体的发现、制备方法、结构类型和设计策略、作用机制以及目前研究现状进行综述。.

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus(HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.

The coordinated recognition of virus-derived T cell epitopes and MHC molecules by T cells plays a pivotal role in cellular immunity–mediated virus clearance. It has been demonstrated that the conformation of MHC class I (MHC I) molecules can be adjusted by the presented peptide, which impacts T cell activation. However, it is still largely unknown whether the conformational shift of MHC I influences the protective effect of virus-specific T cells. In this study, utilizing the Middle East respiratory syndrome coronavirus–infected mouse model, we observed that through the unusual secondary anchor Ile5, a CD8+ T cell epitope drove the conformational fit of Trp73 on the α1 helix of murine MHC I H-2Kd. In vitro renaturation and circular dichroism assays indicated that this shift of the structure did not influence the peptide/MHC I binding affinity. Nevertheless, the T cell recognition and the protective effect of the peptide diminished when we made an Ile to Ala mutation at position 5 of the original peptide. The molecular bases of the concordant recognition of T cell epitopes and host MHC-dependent protection were demonstrated through both crystal structure determination and tetramer staining using the peptide–MHC complex. Our results indicate a coordinated MHC I/peptide interaction mechanism and provide a beneficial reference for T cell–oriented vaccine development against emerging viruses such as Middle East respiratory syndrome coronavirus.

The commonly used Bacillus Calmette-Guérin (BCG) vaccine only induces moderate T cell responses and is less effective in protecting against pulmonary tuberculosis (TB) in adults and ageing populations. Thus, developing new TB vaccine candidates is an important strategy against the spread of Mycobacterium tuberculosis. Here, we demonstrated that immunization with heat-shock protein gp96 as an adjuvant led to a significantly increased CD4(+) and CD8(+) T cell response to a BCG vaccine. Secretion of the Th1-type cytokines was increased by splenocytes from gp96-immunized mice. In addition, adding gp96 as an adjuvant effectively improved the protection against intravenous challenge with Mycobacterium bovis BCG in mice. Our study reveals the novel property of gp96 in boosting the vaccine-specific T cell response and its potential use as an adjuvant for BCG vaccines against mycobacterial infection.

Defining and loading of immunogenic and safe cancer antigens remain a major challenge for designing dendritic cell (DC)-based cancer vaccines. In this study, we defined a prototype strategy of using DC-based vaccines pulsed with placenta-derived heat shock protein gp96 to induces anti-tumor T cell responses. Placental gp96 was efficiently taken up by CD11c+ bone marrow-derived DCs (BMDCs) and resulted in moderate BMDC maturation. Splenocytes and cytotoxic T cells (CTLs) generated with mouse BMDCs pulsed with placental gp96 specifically lysed B16 melanoma and LLC lung carcinoma cells. In both transplantable melanoma and lung carcinoma mice models, immunization with placental gp96-stimulated BMDCs led to a significant decrease in tumor growth and mouse mortality with respect to mice treated with liver gp96-pulsed BMDCs or placental gp96 alone. This vaccine induced strong cross-reactive tumor-specific T cell responses. Our results revealed that DCs pulsed with placenta-derived gp96 represent an effective immunotherapy to induce tumor-reactive immune responses, possibly via loading DCs with its associated carcinoembryonic antigens.

Under the immune pressure of cytotoxic T cells (CTLs), hepatitis B virus (HBV) evolves to accumulate mutations more likely within epitopes to evade immune detection. However, little is known about the specific patterns of the immune pressure-associated HBV mutation of T-cell epitopes and their link to disease progression. Here, we observed a correlation of the accumulated variants on HBV core protein (HBc) with the disease severity of HBV infection. Further analysis indicated that these substitutions were mostly located within CD8+ T-cell epitopes of HBc protein, which were systematically screened and identified in an unbiased manner in our study. From individual peptide level to the human leukocyte antigen I (HLA-I)-restricted population level, we elucidated that the mutations in these well-defined HLA-I-restricted T-cell epitopes significantly decreased antiviral activity-specific CTLs and were positively associated with clinical parameters and disease progression in HBV-infected patients. The molecular pattern for viral epitope variations based on the sequencing of 105 HBV virus genomes indicated that the C-terminal portion (Pc), especially the Pc-1 and Pc-2 positions, have the highest mutation rates. Further structural analysis of HLA-A*02 complexed to diverse CD8+ T-cell epitopes revealed that the highly variable C-terminal bulged peak of M-shaped HBc-derived epitopes are solvent exposed, and most of the CDR3βs of the T-cell receptor hover over them. These data shed light on the molecular and immunological mechanisms of T-cell immunity-associated viral evolution in hepatitis B progression, which is beneficial for designing immunotherapies and vaccines.IMPORTANCE The specific patterns of sequence polymorphisms of T-cell epitopes and the immune mechanisms of the HBV epitope mutation-linked disease progression are largely unclear. In this study, we systematically evaluated the contribution of CD8+ T cells to the disease progress-associated evolution of HBV. By evaluation of patient T-cell responses based on the peptide repertoire, we comprehensively characterized the association of clinical parameters in chronic hepatitis B with the antiviral T-cell response-associated mutations of the viruses from the single-epitope level to the overall HLA-I-restricted peptide levels. Furthermore, we investigated the molecular basis of the HLA-A2-restricted peptide immune escape and found that the solvent-exposed C-terminal portion of the epitopes is highly variable under CDR3β recognition. Our work may provide a comprehensive evaluation of viral mutations impacted by the host CTL response in HBV disease progression in the context of the full repertoire of HBc-derived epitopes.

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.

The resistance to apoptosis displayed by liver cancer plays a key role in hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-therapy. In this study, we uncovered the potential role and mechanism of heat shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53 protein was identified as a gp96 client protein by profiling apoptosis-related proteins in gp96-knockdown liver cancer cells. Overexpression and knockdown studies both demonstrated that gp96 decreases p53 protein levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We further provide evidence that gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induced cell apoptosis and led to the suppression of liver tumor growth in vivo. In conclusion, we elucidated an underlying mechanism by which gp96 promotes p53 degradation via increasing Mdm2 E3 ligase activity and provided a new therapeutic strategy to target the gp96-mediated anti-apoptotic characteristic of hepatocellular carcinoma.

Figure S5. Reciprocal ceRNA activity between HER2 and HER3 3â UTR. HER2 3â UTR-luciferase reporter assay in T47D cells transfected with the HER3 3â UTR or control vector. (PDF 150 kb)

Figure S3. Combinatory treatment with HER3 siRNA and trastuzumab is useful for overcoming trastuzumab resistance. (A-C) Real-time PCR (A), western blotting (B), and FACS (C) analysis of HER2 and HER3 expression in AU565 parental and trastuzumab-resistant (TtzmR) cell lines. (D) Proliferation of AU565 parental and TtzmR cells treated with 10 μg/ml trastuzumab or control IgG, along with a cholesterol-conjugated siRNA targeting HER3 or a randomized oligonucleotide (control). All error bars represent the standard deviation. All quantitative data were generated from a minimum of three replicates. (E, F) AU565 TtzmR cells were s.c. injected into female BALB/c-nude mice. Mice were treated with cholesterol-conjugated HER3 siRNA or trastuzumab at days 0, 7, and 14. Representative in vivo luciferase images of mice at days 0, 10, and 21(E). The results are presented as means ± SD from five mice. Immunostaining of HER3 in xenograft tumor sections (F). Red, HER3; Blue, DAPI. Scale bar, 40 μm. (PDF 3149 kb)

Figure S2. Effect of trastuzumab on HER2 and HER3 levels. FACS analysis of HER2 and HER3 levels in AU565 cells treated with 10Â Îźg/ml trastuzumab at the indicated times or the indicated concentrations of trastuzumab. (PDF 1027 kb)

Figure S1. Effect of trastuzumab on miR-125a/b levels. Real-time PCR analysis of miR-125a and miR-125b levels in AU565 cells treated with 10Â Îźg/ml trastuzumab at the indicated times. Data were generated from three replicates. (PDF 295 kb)

Figure S4. Correlation between miR-125a/b and EGFR family proteins in HER2 positive breast cancer patients. Correlation between miR-125a and miR-125b and EGFR family proteins (EGFR, HER2 and HER3) in HER2 positive breast cancer patients. (PDF 1450 kb)