搜索结果: 'gmp t cell activator'

The T-cell surface molecule TIGIT is an immune checkpoint molecule that inhibits T-cell responses,but its roles in cancer are little understood. In this study,we evaluated the role TIGIT checkpoint plays in the development and progression of gastric cancer. We show that the percentage of CD8 T cells that are TIGIT+ was increased in gastric cancer patients compared with healthy individuals. These cells showed functional exhaustion with impaired activation,proliferation,cytokine production,and metabolism,all of which were rescued by glucose. In addition,gastric cancer tissue and cell lines expressed CD155,which bound TIGIT receptors and inactivated CD8 T cells. In a T cell-gastric cancer cell coculture system,gastric cancer cells deprived CD8 T cells of glucose and impaired CD8 T-cell effector functions; these effects were neutralized by the additional glucose or by TIGIT blockade. In gastric cancer tumor cells,CD155 silencing increased T-cell metabolism and IFNγ production,whereas CD155 overexpression inhibited T-cell metabolism and IFNγ production; this inhibition was neutralized by TIGIT blockade. Targeting CD155/TIGIT enhanced CD8 T-cell reaction and improved survival in tumor-bearing mice. Combined targeting of TIGIT and PD-1 further enhanced CD8 T-cell activation and improved survival in tumor-bearing mice. Our results suggest that gastric cancer cells inhibit CD8 T-cell metabolism through CD155/TIGIT signaling,which inhibits CD8 T-cell effector functions,resulting in hyporesponsive antitumor immunity. These findings support the candidacy of CD155/TIGIT as a potential therapeutic target in gastric cancer. Cancer Res; 77(22); 6375-88. textcopyright2017 AACR. View Publication

PURPOSE Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors,such as prostate cancers. However,targeting MDSCs proved challenging due to their phenotypic heterogeneity. EXPERIMENTAL DESIGN Myeloid cell populations were evaluated using flow cytometry on blood samples,functional assays,and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects,localized and metastatic castration-resistant prostate cancer patients. RESULTS Here,we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3,which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA,thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1,a downstream STAT3 target gene and a potent T-cell inhibitor. CONCLUSIONS Overall,we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression. View Publication

BACKGROUND Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10{\%} DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry,as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel,each sample was flow sorted into fibroblast,T-cell,B-cell,and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS Upon dissociation,cryopreserved synovial tissue fragments yielded a high frequency of viable cells,comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with {\~{}} 30 arthroplasty and {\~{}} 20 biopsy samples yielded a consensus digestion protocol using 100 mu$g/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes,distinct populations of memory B cells and antibody-secreting cells,and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes,fibroblasts,and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell,including transcripts encoding characteristic lineage markers identified. CONCLUSIONS We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. View Publication

Recent advances in the differentiation and production of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) have stimulated development of strategies to use these cells in human cardiac regenerative therapies. A prerequisite for clinical trials and translational implementation of hPSC-derived CMs is the ability to manufacture safe and potent cells on the scale needed to replace cells lost during heart disease. Current differentiation protocols generate fetal-like CMs that exhibit proarrhythmogenic potential. Sufficient maturation of these hPSC-derived CMs has yet to be achieved to allow these cells to be used as a regenerative medicine therapy. Insights into the native cardiac environment during heart development may enable engineering of strategies that guide hPSC-derived CMs to mature. Specifically,considerations must be made in regard to developing methods to incorporate the native intercellular interactions and biomechanical cues into hPSC-derived CM production that are conducive to scale-up. View Publication

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material,the mouse whipwormTrichuris murishas been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products ofT. muris. We identify 148 proteins secreted byT. murisand show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep{\textregistered} gradient to purify the EVs,highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs,identifying {\textgreater}350 proteins,56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping toT. murisgene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation,implying a role in parasite-driven immunomodulation. In addition,for the first time to our knowledge,colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted byT. murisprovides important information on whipworm-host communication and forms the basis for future studies. View Publication

We hypothesized that the neonatal rat heart would bring transplanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to maturity as it grows to adult size. In neonatal rat heart,engrafted hiPSC derivatives developed partially matured myofibrils after 3 months,with increasing cell size and sarcomere length. There was no difference between grafts from hiPSC-CMs or hiPSC-derived cardiac progenitors (hiPSC-CPs) at 3 months,nor was maturation influenced by infarction. Interestingly,the infarcted adult heart induced greater human cardiomyocyte hypertrophy and induction of cardiac troponin I expression than the neonatal heart. Although human cardiomyocytes at all time points were significantly smaller than the host rat cardiomyocytes,transplanted neonatal rat cardiomyocytes reached adult size and structure by 3 months. Thus,the adult rat heart induces faster maturation than the neonatal heart,and human cardiomyocytes mature more slowly than rat cardiomyocytes. The slower maturation of human cardiomyocytes could be related to environmental mismatch or cell-autonomous factors. View Publication

Enteric pathogens including Salmonella enteric serovar Typhimurium can breach the epithelial barrier of the host and spread to systemic tissues. In response to infection,the host activates innate immune receptors via the signaling molecule MyD88,which induces protective inflammatory and antimicrobial responses. Most of these innate immune responses have been studied in hematopoietic cells,but the role of MyD88 signaling in other cell types remains poorly understood. Surprisingly,we found that Dermo1-Cre;Myd88fl/fl mice with mesenchymal cell-specific deficiency of MyD88 were less susceptible to orogastric and i.p. STyphimurium infection than their Myd88fl/fl littermates. The reduced susceptibility of Dermo1-Cre;Myd88fl/fl mice to infection was associated with lower loads of S. Typhimurium in the liver and spleen. Mutant analyses revealed that S. Typhimurium employs its virulence type III secretion system 2 to promote its growth through MyD88 signaling pathways in mesenchymal cells. Inflammatory monocytes function as a major cell population for systemic dissemination of S. Typhimurium Mechanistically,mesenchymal cell-specific MyD88 signaling promoted CCL2 production in the liver and spleen and recruitment of inflammatory monocytes to systemic organs in response to STyphimurium infection. Consistently,MyD88 signaling in mesenchymal cells enhanced the number of phagocytes including Ly6ChiLy6G- inflammatory monocytes harboring STyphimurium in the liver. These results suggest that S. Typhimurium promotes its systemic growth and dissemination through MyD88 signaling pathways in mesenchymal cells. View Publication

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations,since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina,it may be possible to use one cell type to rescue another cell type in the face of severe stress,such as hypoxia or genetically encoded cell-specific degenerations. Here,we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore,we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44,the hyaluronic acid receptor,and IGFBPs (insulin-like growth factor-binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases. View Publication

Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells,but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinal lamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium,and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5+ intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced,whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens. View Publication