技术资料

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here,we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP,termed the wing region. One mAb targeting the GP2 wing,MR228,showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb,MR235,to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs,resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection. View Publication

Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely,how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK),which is blocked with a selective p38$\alpha$/$\beta$ MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically,we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether,these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation. View Publication

The inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However,the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here,we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological,immunological,and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions,however,viral replication and the clinical course of infection are attenuated. Thus,Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion. View Publication

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells,T cells,and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75{\%} of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7,the ocular T cell population increases to 50{\%} of CD45 + cells,leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible ({\textless} 1{\%} of CD45 + cells),the ocular B cell population increases to {\textgreater} 4{\%} by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models. View Publication

Recent developments in understanding how the functional phenotype of the innate immune system is programmed has led to paradigm-shifting views on immunomodulation. These advances have overturned two long-held dogmas: (1) only adaptive immunity confers immunological memory; and,(2) innate immunity lacks specificity. This work describes the observation that innate immune effector cells appear to be differentially recruited to specific pathological sites when mobilized by distinct inactivated bacterial-based stimuli administered subcutaneously. The studies presented suggest that the immune system,upon detecting the first signs of a potential infection by a specific pathogen,tends to direct its resources to the compartment from which that pathogen is most likely originating. The findings from this work puts forth the novel hypothesis that the immunotherapeutic efficacy of a microbial-based stimulus for innate immune mobilization depends on the correct selection of the microbial species used as the stimulant and its relationship to the organ in which the pathology is present. View Publication

Interferon regulatory factor 1 (IRF1) regulates diverse biological functions,including modulation of cellular responses involved in tumorigenesis. Genetic mutations and altered IRF1 function are associated with several cancers. Although the function of IRF1 in the immunobiology of cancer is emerging,IRF1-specific mechanisms regulating tumorigenesis and tissue homeostasis in vivo are not clear. Here,we found that mice lacking IRF1 were hypersusceptible to colorectal tumorigenesis. IRF1 functions in both the myeloid and epithelial compartments to confer protection against AOM/DSS-induced colorectal tumorigenesis. We further found that IRF1 also prevents tumorigenesis in a spontaneous mouse model of colorectal cancer. The attenuated cell death in the colons of Irf1-/- mice was due to defective pyroptosis,apoptosis,and necroptosis (PANoptosis). IRF1 does not regulate inflammation and the inflammasome in the colon. Overall,our study identified IRF1 as an upstream regulator of PANoptosis to induce cell death during colitis-associated tumorigenesis. View Publication

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present,non-invasive liquid (blood) biopsy is not utilized for leukemia,due to lower counts of leukemia blast cells in the blood. Here,we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system,without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards,such as flow cytometry,using BM aspirates. Blast cell detection efficiency was higher in 55.6{\%} of the patients using the BCB as compared to flow cytometry,despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients. View Publication

The process of oligodendrogenesis has been relatively well delineated in the rodent brain. However,it remains unknown whether analogous developmental processes are manifested in the human brain. Here we report oligodendrogenesis in forebrain organoids,generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) versus dorsal forebrain organoids (DFOs). Interestingly,oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore,dorsally derived oligodendroglial cells outcompete ventrally derived oligodendroglia and become dominant in FFOs after long-term culture. Thus,our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally and dorsally derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects. View Publication

Thymosin $\beta$4 (T$\beta$4) is a G-actin sequestering protein that contributes to diverse cellular activities,such as migration and angiogenesis. In this study,the beneficial effects of combined cell therapy with T$\beta$4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with T$\beta$4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly,T$\beta$4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with T$\beta$4 significantly increased cell migration and sprouting from microbeads. Moreover,additional treatment with T$\beta$4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the T$\beta$4-hASCs combination on vessel recruitment,dorsal window chambers were transplanted,and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with T$\beta$4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together,the therapeutic application of hASCs combined with T$\beta$4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia. View Publication

Chronic lymphocytic leukaemia (CLL) exhibits differences between Asians and Caucasians in terms of incidence rate,age at onset,immunophenotype,and genetic profile. We performed genome-wide methylation profiling of CLL in an Asian cohort for the first time. Eight Korean patients without somatic immunoglobulin heavy chain gene hypermutations underwent methyl-CpG-binding domain sequencing (MBD-seq),as did five control subjects. Gene Ontology,pathway analysis,and network-based prioritization of differentially methylated genes were also performed. More regions were hypomethylated (2,062 windows) than were hypermethylated (777 windows). Promoters contained the highest proportion of differentially methylated regions (0.08{\%}),while distal intergenic and intron regions contained the largest number of differentially methylated regions. Protein-coding genes were the most abundant,followed by long noncoding and short noncoding genes. The most significantly over-represented signalling pathways in the differentially methylated gene list included immune/cancer-related pathways and B-cell receptor signalling. Among the top 10 hub genes identified via network-based prioritization,four (UBC,GRB2,CREBBP,and GAB2) had no known relevance to CLL,while the other six (STAT3,PTPN6,SYK,STAT5B,XPO1,and ABL1) have previously been linked to CLL in Caucasians. As such,our analysis identified four novel candidate genes of potential significance to Asian patients with CLL. View Publication

PURPOSE Patients with metastatic colorectal cancer refractory to chemotherapy have limited treatment options. Ensituximab (NEO-102) is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer. PATIENTS AND METHODS Single-arm,phase II trial assessed the efficacy and safety of ensituximab in patients with advanced,refractory cancer who expressed MUC5AC antigen in tumor tissue. Ensituximab was administered intravenously every 2 weeks with 3 mg/kg as recommended phase II dose (RP2D). A minimum sample size of 43 patients was required on the basis of the assumption that ensituximab would improve median overall survival (OS) by 7 months using a one-sided significance level of 10{\%} and 80{\%} power. Written informed consent was obtained from all patients. RESULTS Sixty-three patients with advanced,refractory colorectal cancer were enrolled and 53 subjects were treated in phase II arm. Median age was 58 years and 46{\%} of the patients were female. Among 57 evaluable patients,median OS was 6.8 months. No responses were observed,and stable disease was achieved in 21{\%} of the patients. The most common treatment-related adverse events (AE) at RP2D included fatigue (38{\%}),anemia (30{\%}),nausea (15{\%}),vomiting (11{\%}),increased bilirubin (9{\%}),constipation (8{\%}),decreased appetite (6{\%}),and diarrhea (6{\%}). Serious AEs at least possibly related to ensituximab occurred in 4 patients and included anemia,nausea,increased bilirubin,and hypoxia. No patients discontinued treatment due to drug-related AEs. CONCLUSIONS Ensituximab was well tolerated and demonstrated modest antitumor activity in patients with heavily pretreated refractory colorectal cancer. View Publication

Much of our understanding of chromosome segregation is based on cell culture systems. Here,we examine the importance of the tissue environment for chromosome segregation by comparing chromosome segregation fidelity across several primary cell types in native and nonnative contexts. We discover that epithelial cells have increased chromosome missegregation outside of their native tissues. Using organoid culture systems,we show that tissue architecture,specifically integrin function,is required for accurate chromosome segregation. We find that tissue architecture enhances the correction of merotelic microtubule-kinetochore attachments,and this is especially important for maintaining chromosome stability in the polyploid liver. We propose that disruption of tissue architecture could underlie the widespread chromosome instability across epithelial cancers. Moreover,our findings highlight the extent to which extracellular context can influence intrinsic cellular processes and the limitations of cell culture systems for studying cells that naturally function within a tissue. Tissue architecture and integrin function are critical factors that support chromosome segregation fidelity in epithelial tissues. View Publication