技术资料

Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3$\zeta$ or 4-1BB/CD3$\zeta$ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function,clinical efficacy,and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3$\zeta$ and 4-1BB/CD3$\zeta$ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3$\zeta$ CARs activated faster and larger-magnitude changes in protein phosphorylation,which correlated with an effector T cell-like phenotype and function. In contrast,4-1BB/CD3$\zeta$ CAR T cells preferentially expressed T cell memory-associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3$\zeta$ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus,tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity. View Publication

Immunomodulation contributes to the antitumor efficacy of the fractionated radiation therapy (RT). Here,we describe immune effects of RT with concurrent systemic cisplatin or cetuximab treatment of patients with stage III-IV head and neck squamous cell carcinoma (HNSCC). Using longitudinally collected blood samples,we identified significant changes in cytokines/chemokines and immune cell populations compared to immune-related gene expression profiles in peripheral blood mononuclear cells (PBMCs). The 7-week combinatorial RT resulted in gradual elevation of proinflammatory mediators (IFNgamma$,IL-6,TNFɑ,CCL2),while levels of IL-12,cytokine essential for antitumor immune responses,were decreased. These effects correlated with progressive accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) with detectable activity of STAT3 and PD-L1 expression,underscoring tolerogenic effects of MDSCs. Correspondingly,gene expression analysis of PBMCs harvested after two weeks of combinatorial RT,found upregulation of several immunosuppressive mediators. These included IL6,IL6R,STAT3 and PDL1,which could represent IL-6/STAT3-driven tolerogenic signaling,which inhibits T cell and NK activity. Overall,our results suggest that potential immunostimulatory effects of combinatorial RT in HNSCC patients are likely limited by tolerogenic STAT3 signaling and PD-L1 upregulation in myeloid immune cells. Further studies will clarify whether STAT3 targeting could augment RT efficacy and durability of antitumor responses. 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

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

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

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

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

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 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

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported,current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here,we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs.backslashnbackslashnMETHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs,an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes,a mouse cardiomyocyte cell line,but textless3% of 4 noncardiomyocyte cell types in flow cytometry analysis,which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes,which supports the specificity of MBs. Finally,MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures,and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics,which was verified by spontaneous beating,electrophysiological studies,and expression of cardiac proteins. When transplanted in a myocardial infarction model,MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.backslashnbackslashnCONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery. View Publication

BACKGROUND Triple-negative breast cancer (TNBC) has significantly worse prognosis. Acquired chemoresistance remains the major cause of therapeutic failure of TNBC. In clinic,the relapsed TNBC is commonly pan-resistant to various drugs with completely different resistant mechanisms. Investigation of the mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients. METHODS In this study,1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT),combination index (CI)-isobologram,western blot,ALDEFLUOR analysis,clonogenic assay and immunocytochemistry were used. RESULTS The chemoresistant MDA-MB-231PAC10 cells are highly cross-resistant to paclitaxel (PAC),cisplatin (CDDP),docetaxel and doxorubicin. The MDA-MB-231PAC10 cells are quiescent with significantly longer doubling time (64.9 vs 31.7 h). This may be caused by high expression of p21(Waf1). The MDA-MB-231PAC10 cells express high aldehyde dehydrogenase (ALDH) activity and a panel of embryonic stem cell-related proteins,for example,Oct4,Sox2,Nanog and nuclealisation of HIF2$$ and NF-$$Bp65. We have previously reported that disulfiram (DS),an antialcoholism drug,targets cancer stem cells (CSCs) and enhances cytotoxicity of anticancer drugs. Disulfiram abolished CSC characters and completely reversed PAC and CDDP resistance in MDA-MB-231PAC10 cells. CONCLUSION Cancer stem cells may be responsible for acquired pan-chemoresistance. As a drug used in clinic,DS may be repurposed as a CSC inhibitor to reverse the acquired pan-chemoresistance. View Publication

RATIONALE: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.backslashnbackslashnOBJECTIVE: To test the hypothesis that a short-course,dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.backslashnbackslashnMETHODS AND RESULTS: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein,and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury,as assessed by magnetic resonance imaging. Mechanistically,costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile.backslashnbackslashnCONCLUSIONS: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment,costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism. View Publication