技术资料

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study,we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine),and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses,antigen-specific proliferation of peripheral blood mononuclear cells,gross and microscopic lung lesions,and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8$\alpha$$\alpha$+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition,pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically,increased IFN-$\gamma$ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However,in the KAg nanovaccine-immunized pigs,hemagglutination inhibition,IgG and IgA antibody responses,and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall,our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs,with promise to induce cross-protective immunity. View Publication

Immunotherapy has increased overall survival of metastatic cancer patients,and cancer antigens are promising vaccine targets. To fulfill the promise,appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice,and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart,we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC,both important in human cancer development and progression,were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09,CASAC,Montanide ISA 51 VG,or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs),the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01,-1(*)07:02,-2(*)04:01,-2(*)05:02,and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-$\gamma$ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses,and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer. View Publication

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system,we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes,including TMPRSS2-ERG fusion,SPOP mutation,SPINK1 overexpression,and CHD1 loss. Whole-exome sequencing shows a low mutational burden,consistent with genomics studies,but with mutations in FOXA1 and PIK3R1,as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. View Publication

Small-cell lung cancer (SCLC),an aggressive neuroendocrine tumor with early dissemination and dismal prognosis,accounts for 15-20{\%} of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable,and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs),which are prevalent in SCLC,present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice,and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged,and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms. View Publication

Diverse hematopoietic progenitors,including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells,mobilized by hematopoietic growth factors or emerging during parasitic infections,display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-$\gamma$ released by activated effector T cells (Teffs),by up-regulating their Fas ligand (FasL) expression,which enabled them to kill Teffs through apoptosis. In turn,IFN-$\gamma$ derived from CpG-proBs enhanced IFN-$\gamma$ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D,adoptively transferred IFN-$\gamma$-deficient CpG-proBs did not prevent T1D development. Additionally,CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation. View Publication

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However,the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular,interferon-gamma$ (IFN-gamma$)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-gamma$ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation,the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore,the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-gamma$ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-gamma$ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture,whereas IDO expression by IFN-gamma$-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore,sustained IDO expression induced by IFN-gamma$-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-gamma$-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether,microparticle delivery of IFN-gamma$ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237. View Publication

Granulocyte colony-stimulating factor (G-CSF or CSF3) and its receptor CSF3R regulate granulopoiesis,neutrophil function,and hematopoietic stem cell mobilization. Recent studies have uncovered an oncogenic role of mutations in the CSF3R gene in many hematologic malignancies. To find additional CSF3R mutations that give rise to cell transformation,we performed a cellular transformation assay in which murine interleukin 3 (IL-3)-dependent Ba/F3 cells were transduced with WT CSF3R plasmid and screened for spontaneous growth in the absence of IL-3. Any outgrowth clones were sequenced to identify CSF3R mutations with transformation capacity. We identified several novel mutations and determined that they transform cells via four distinct mechanisms: 1) cysteine- and disulfide bond-mediated dimerization (S581C); 2) polar,noncharged amino acid substitution at the transmembrane helix dimer interface at residue Thr-640; 3) increased internalization by a Glu-524 substitution that mimics a low G-CSF dose; and 4) hydrophobic amino acid substitutions in the membrane-proximal residues Thr-612,Thr-615,and Thr-618. Furthermore,the change in signaling activation was related to an altered CSF3R localization. We also found that CSF3R-induced STAT3 and ERK activations require CSF3R internalization,whereas STAT5 activation occurred at the cell surface. Cumulatively,we have expanded the regions of the CSF3R extracellular and transmembrane domains in which missense mutations exhibit leukemogenic capacity and have further elucidated the mechanistic underpinnings that underlie altered CSF3R expression,dimerization,and signaling activation. View Publication

The intestinal microbiota has been identified as an environmental factor that markedly affects energy storage and body-fat accumulation in mammals,yet the underlying mechanisms remain unclear. Here we show that the microbiota regulates body composition through the circadian transcription factor NFIL3.Nfil3transcription oscillates diurnally in intestinal epithelial cells,and the amplitude of the circadian oscillation is controlled by the microbiota through group 3 innate lymphoid cells,STAT3 (signal transducer and activator of transcription 3),and the epithelial cell circadian clock. NFIL3 controls expression of a circadian lipid metabolic program and regulates lipid absorption and export in intestinal epithelial cells. These findings provide mechanistic insight into how the intestinal microbiota regulates body composition and establish NFIL3 as an essential molecular link among the microbiota,the circadian clock,and host metabolism. View Publication

Adequate availability of cellular building blocks,including lipids,is a prerequisite for cellular proliferation,but excess dietary lipids are linked to increased cancer risk. Despite these connections,specific regulatory relationships between membrane composition,intestinal stem cell (ISC) proliferation,and tumorigenesis are unclear. We reveal an unexpected link between membrane phospholipid remodeling and cholesterol biosynthesis and demonstrate that cholesterol itself acts as a mitogen for ISCs. Inhibition of the phospholipid-remodeling enzyme Lpcat3 increases membrane saturation and stimulates cholesterol biosynthesis,thereby driving ISC proliferation. Pharmacologic inhibition of cholesterol synthesis normalizes crypt hyperproliferation in Lpcat3-deficient organoids and mice. Conversely,increasing cellular cholesterol content stimulates crypt organoid growth,and providing excess dietary cholesterol or driving endogenous cholesterol synthesis through SREBP-2 expression promotes ISC proliferation in vivo. Finally,disruption of Lpcat3-dependent phospholipid and cholesterol homeostasis dramatically enhances tumor formation in Apcminmice. These findings identify a critical dietary-responsive phospholipid-cholesterol axis regulating ISC proliferation and tumorigenesis. View Publication

BACKGROUND Patient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However,the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research,and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study,our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer. METHODS We employed organoid culture methods and flow cytometric,cytologic,immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment. RESULTS We describe the culture and characterization of human pancreatic cancer organoids from resection,ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology,tissue architecture and polarity in contrast to cell line spheroids,which formed homogenous,non-lumen forming spheres. Importantly,we demonstrate the construction of complex organotypic models of tumor,stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models. CONCLUSIONS These studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor,stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration. View Publication

Cells demonstrate plasticity following injury,but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing,we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically,SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly,we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways,they may serve as a reserve cell source for enhancing human airway regeneration. View Publication

The tryptophan metabolite kynurenine has critical immunomodulatory properties and can function as an aryl hydrocarbon receptor (AHR) ligand. Here we show that the ability of T cells to transport kynurenine is restricted to cells activated by the T-cell antigen receptor or proinflammatory cytokines. Kynurenine is transported across the T-cell membrane by the System L transporter SLC7A5. Accordingly,the ability of kynurenine to activate the AHR is restricted to T cells that express SLC7A5. We use the fluorescence spectral properties of kynurenine to develop a flow cytometry-based assay for rapid,sensitive and quantitative measurement of the kynurenine transport capacity in a single cell. Our findings provide a method to assess the susceptibility of T cells to kynurenine,and a sensitive single cell assay to monitor System L amino acid transport. View Publication