技术资料

During viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi's sarcoma-associated herpesvirus interacts and inhibits the central cell death effector,Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins,like vFlip,on liver physiology in vivo,mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level,hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably,embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice,culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis. View Publication

Seasonal and pandemic influenza infection remains a major public health concern worldwide. Driving robust humoral immunity has been a challenge given preexisting,often cross-reactive,immunity and in particular,poorly immunogenic avian antigens. To overcome immune barriers,the adjuvant MF59 has been used in seasonal influenza vaccines to increase antibody titers and improve neutralizing activity,translating to a moderate increase in protection in vulnerable populations. However,its effects on stimulating antibody effector functions,including NK cell activation,monocyte phagocytosis,and complement activity,all of which have been implicated in protection against influenza,have yet to be defined. Using systems serology,we assessed changes in antibody functional profiles in individuals who received H5N1 avian influenza vaccine administered with MF59,with alum,or delivered unadjuvanted. MF59 elicited antibody responses that stimulated robust neutrophil phagocytosis and complement activity. Conversely,vaccination with MF59 recruited NK cells poorly and drove moderate monocyte phagocytic activity,both likely compromised because of the induction of antibodies that did not bind FCGR3A. Collectively,defining the humoral antibody functions induced by distinct adjuvants may provide a path to designing next-generation vaccines that can selectively leverage the humoral immune functions,beyond binding and neutralization,resulting in better protection from infection. View Publication

Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments,better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children's Oncology Group ACNS02B3 study,we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology,immunohistochemistry,gene expression,DNA methylation,copy number,and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies. View Publication

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs),though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms,including platelet activation and neutrophil-extracellular trap formation; therefore,an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here,we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC),a sulfhydryl-containing compound with broad effects on glutathione replenishment,free radical scavenging,and reducing disulfide bonds,to investigate its antithrombotic effects in the context of MPN. Strikingly,NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo,we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore,NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN. View Publication

The placenta and umbilical cord are pre-eminent candidate sources of mesenchymal stem cells (MSCs). However,placenta-derived MSCs (P-MSCs) showed greater proliferation capacity than umbilical cord-derived MSCs (UC-MSCs) in our study. We investigated the drivers of this proliferation difference and elucidated the mechanisms of proliferation regulation. Proteomic profiling and Gene Ontology (GO) functional enrichment were conducted to identify candidate proteins that may influence proliferation. Using lentiviral or small interfering RNA infection,we established overexpression and knockdown models and observed changes in cell proliferation to examine whether a relationship exists between the candidate proteins and proliferation capacity. Real-time quantitative polymerase chain reaction,western blot analysis,and immunofluorescence assays were conducted to elucidate the mechanisms underlying proliferation. Six candidate proteins were selected based on the results of proteomic profiling and GO functional enrichment. Through further validation,yes-associated protein 1 (YAP1) and $\beta$-catenin were confirmed to affect MSCs proliferation rates. YAP1 and $\beta$-catenin showed increased nuclear colocalization during cell expansion. YAP1 overexpression significantly enhanced proliferation capacity and upregulated the expression of both $\beta$-catenin and the transcriptional targets of Wnt signaling,CCND1,and c-MYC,whereas silencing $\beta$-catenin attenuated this influence. We found that YAP1 directly interacts with $\beta$-catenin in the nucleus to form a transcriptional YAP/$\beta$-catenin/TCF4 complex. Our study revealed that YAP1 and $\beta$-catenin caused the different proliferation capacities of P-MSCs and UC-MSCs. Mechanism analysis showed that YAP1 stabilized the nuclear $\beta$-catenin protein,and also triggered the Wnt/$\beta$-catenin pathway,promoting proliferation. View Publication

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health,with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom,phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding ({\textgreater}103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium,it is the relatively rapid and higher levels of Stx2a induction,compared to Stx2c,that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium. View Publication

Here,we report on the results of a phase I/II trial (NCT00490529) for patients with mantle cell lymphoma who,having achieved remission after immunochemotherapy,were vaccinated with irradiated,CpG-activated tumor cells. Subsequently,vaccine-primed lymphocytes were collected and reinfused after a standard autologous stem cell transplantation (ASCT). The primary endpoint was detection of minimal residual disease (MRD) within 1 yr after ASCT at the previously validated threshold of ≥1 malignant cell per 10,000 leukocyte equivalents. Of 45 evaluable patients,40 (89{\%}) were found to be MRD negative,and the MRD-positive patients experienced early subsequent relapse. The vaccination induced antitumor CD8 T cell immune responses in 40{\%} of patients,and these were associated with favorable clinical outcomes. Patients with high tumor PD-L1 expression after in vitro exposure to CpG had inferior outcomes. Vaccination with CpG-stimulated autologous tumor cells followed by the adoptive transfer of vaccine-primed lymphocytes after ASCT is feasible and safe. View Publication

The Glioblastoma (GBM) immune microenvironment plays a critical role in tumor development,progression,and prognosis. A comprehensive understanding of the intricate milieu and its interactions remains unclear,and single-cell analysis is crucially needed. Leveraging mass cytometry (CyTOF),we analyzed immunocytes from 13 initial and three recurrent GBM samples and their matched peripheral blood mononuclear cells (pPBMCs). Using a panel of 30 markers,we provide a high-dimensional view of the complex GBM immune microenvironment. Hematoxylin and eosin staining and polychromatic immunofluorescence were used for verification of the key findings. In the initial and recurrent GBMs,glioma-associated microglia/macrophages (GAMs) constituted 59.05 and 27.87{\%} of the immunocytes,respectively; programmed cell death-ligand 1 (PD-L1),T cell immunoglobulin domain and mucin domain-3 (TIM-3),lymphocyte activation gene-3 (LAG-3),interleukin-10 (IL-10) and transforming growth factor-$\beta$ (TGF$\beta$) demonstrated different expression levels in the GAMs among the patients. GAMs could be subdivided into different subgroups with different phenotypes. Both the exhausted T cell and regulatory T (Treg) cell percentages were significantly higher in tumors than in pPBMCs. The natural killer (NK) cells that infiltrated into the tumor lesions expressed higher levels of CXC chemokine receptor 3 (CXCR3),as these cells expressed lower levels of interferon-$\gamma$ (IFN$\gamma$). The immune microenvironment in the initial and recurrent GBMs displayed similar suppressive changes. Our study confirmed that GAMs,as the dominant infiltrating immunocytes,present great inter- and intra-tumoral heterogeneity and that GAMs,increased exhausted T cells,infiltrating Tregs,and nonfunctional NK cells contribute to local immune suppressive characteristics. Recurrent GBMs share similar immune signatures with the initial GBMs except the proportion of GAMs decreases. View Publication

Cyclosporine A (CsA) is a powerful immunosuppressant,but it is an ineffective stand-alone treatment for systemic lupus erythematosus (SLE) due to poor target tissue distribution and renal toxicity. We hypothesized that CD71 (transferrin receptor 1)-directed delivery of CsA to the lymphatic system would improve SLE outcomes in a murine model. We synthesized biodegradable,ligand-conjugated nanoparticles [P2Ns-gambogic acid (GA)] targeting CD71. GA conjugation substantially increased nanoparticle association with CD3+ or CD20+ lymphocytes and with intestinal lymphoid tissues. In orally dosed MRL-lpr mice,P2Ns-GA-encapsulated CsA increased lymphatic drug delivery 4- to 18-fold over the ligand-free formulation and a commercial CsA capsule,respectively. Improved lymphatic bioavailability of CsA was paralleled by normalization of anti-double-stranded DNA immunoglobulin G titer,plasma cytokines,and glomerulonephritis. Thus,this study demonstrates the translational potential of nanoparticles that enhance the targeting of lymphatic tissues,transforming CsA into a potent single therapeutic for SLE. View Publication

Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However,the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1),YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP),a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction,in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally,advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly,gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together,our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy. View Publication

Approaches to deplete persistent HIV infection are needed. We investigated the combined impact of the latency reversing agent vorinostat (VOR) and AGS-004,an autologous dendritic cell immunotherapeutic,on the HIV reservoir. HIV+,stably treated participants in whom resting CD4+ T cell-associated HIV RNA (rca-RNA) increased after VOR exposure ex vivo and in vivo received 4 doses of AGS-004 every 3 weeks,followed by VOR every 72 hours for 30 days,and then the cycle repeated. Change in VOR-responsive host gene expression,HIV-specific T cell responses,low-level HIV viremia,rca-RNA,and the frequency of resting CD4+ T-cell infection (RCI) was measured at baseline and after each cycle. No serious treatment-related adverse events were observed among five participants. As predicted,VOR-responsive host genes responded uniformly to VOR dosing. Following cycles of AGS-004 and VOR,rca-RNA decreased significantly in only two participants,with a significant decrease in SCA observed in one of these participants. However,unlike other cohorts dosed with AGS-004,no uniform increase in HIV-specific immune responses following vaccination was observed. Finally,no reproducible decline of RCI,defined as a decrease of {\textgreater}50{\%},was observed. AGS-004 and VOR were safe and well-tolerated,but no substantial impact on RCI was measured. In contrast to previous clinical data,AGS-004 did not induce HIV-specific immune responses greater than those measured at baseline. More efficacious antiviral immune interventions,perhaps paired with more effective latency reversal,must be developed to clear persistent HIV infection. View Publication