搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

The p110δ isoform of PI3K is known to play an important role in immunity,yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells,we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation,proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases,our study suggests caution in using these drugs in patients,as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens. View Publication

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here,we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF,a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30{\%} engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF,as evidenced by up to 18{\%} HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells,defined by the markers CD34+CD90+CD45RA-,allowing for a 10-fold reduction in the number of transplanted target cells,thus considerably reducing the need for editing reagents. The frequency of engrafted,gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases. View Publication

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM),a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation,we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast,in ML-DS,clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model,we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically,genetically,and transcriptionally mirrored ML-DS. View Publication

The development of effective cancer vaccines remains an urgent,but as yet unmet,clinical need. This deficiency is in part due to an incomplete understanding of how to best invoke dendritic cells (DC) that are crucial for the induction of tumor-specific CD8(+) T cells capable of mediating durable protective immunity. In this regard,elevated expression of the transcription factor X box-binding protein 1 (XBP1) in DC appears to play a decisive role in promoting the ability of DC to cross-present Ags to CD8(+) T cells in the therapeutic setting. Delivery of DNA vaccines encoding XBP1 and tumor Ag to skin DC resulted in increased IFN-? production by plasmacytoid DC (pDC) from skin/tumor draining lymph nodes and the cross-priming of Ag-specific CD8(+) T cell responses associated with therapeutic benefit. Antitumor protection was dependent on cross-presenting Batf3(+) DC,pDC,and CD8(+) T cells. CD103(+) DC from the skin/tumor draining lymph nodes of the immunized mice appeared responsible for activation of Ag-specific naive CD8(+) T cells,but were dependent on pDC for optimal effectiveness. Similarly,human XBP1 improved the capacity of human blood- and skin-derived DC to activate human T cells. These data support an important intrinsic role for XBP1 in DC for effective cross-priming and orchestration of Batf3(+) DC-pDC interactions,thereby enabling effective vaccine induction of protective antitumor immunity. View Publication

Neutrophils (PMNs) contain and release a powerful arsenal of mediators,including several granular enzymes,reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although airway neutrophilia is associated with severity,poor response to glucocorticoids and exacerbations,the pathophysiological role of neutrophils in asthma remains poorly understood. Twenty-four patients with asthma and 22 healthy controls (HCs) were prospectively recruited. Highly purified peripheral blood neutrophils (> 99%) were evaluated for ROS production and activation status upon stimulation with lipopolysaccharide (LPS),N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Plasma levels of myeloperoxidase (MPO),CXCL8,matrix metalloproteinase-9 (MMP-9),granulocyte-monocyte colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF-A) were measured by ELISA. Plasma concentrations of citrullinated histone H3 (CitH3) and circulating free DNA (dsDNA) were evaluated as NET biomarkers. Activated PMNs from asthmatics displayed reduced ROS production and activation status compared to HCs. Plasma levels of MPO,MMP-9 and CXCL8 were increased in asthmatics compared to HCs. CitH3 and dsDNA plasma levels were increased in asthmatics compared to controls and the CitH3 concentrations were inversely correlated to the % decrease in FEV1/FVC in asthmatics. These findings indicate that neutrophils and their mediators could have an active role in asthma pathophysiology. View Publication

Our previous studies have shown that ethanol intoxication combined with burn injury increases intestinal bacterial growth,disrupts the intestinal barrier,and enhances bacterial translocation. Additionally,studies show that Th17 effector cytokines IL-17 and IL-22,which are dependent on IL-23,play important roles in maintaining intestine mucosal barrier integrity. Recent findings suggest neutrophils are a significant source of IL-17 and IL-22. We determined the effect of ethanol and burn injury on neutrophil IL-17 and IL-22 production,as well as their ability to phagocytose and in bacterial clearance,and whether these effects are modulated by IL-23. Mice were given ethanol 4 h prior to receiving ˆ¼12.5% total body surface area burn and were euthanized day 1 after injury. We observed that intoxication combined with burn injury significantly decreases blood neutrophil phagocytosis and bacteria killing,as well as their ability to produce IL-17 and IL-22,compared with sham vehicle mice. The treatment of neutrophils with rIL-23 significantly increases IL-22 and IL-17 release and promotes expression of IL-23R,retinoic acid-related orphan receptor $\gamma$t,Lipocalin2,and Nod-like receptor 2 following ethanol and burn injury. Furthermore,IL-22- and IL-17-producing neutrophils have enhanced neutrophil extracellular trap formation and bacterial killing ability,which are dependent on IL-23. Finally,although we observed that peritoneal neutrophils harvested after casein treatment are functionally different from blood neutrophils,both blood and peritoneal neutrophils exhibited the same response to rIL-23 treatment. Together these findings suggest that IL-23 promotes neutrophil IL-22 and IL-17 production and their ability to kill bacteria following ethanol and burn injury. View Publication

CD8+ T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. Although in vitro-polarized,transferred IL-9-secreting CD8+ Tc9 (cytotoxic T lymphocyte subset 9) cells exert greater persistence and antitumor efficacy than Tc1 cells,the underlying mechanism remains unclear. Here,we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models,which is strongly correlated with their persistence. Using RNA-sequence and functional validation,we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3,upregulated fatty acid oxidation and mitochondrial activity,and rendered Tc9 cells with reduced lipid peroxidation and resistance to tumor- or ROS-induced ferroptosis in the tumor microenvironment. IL-9 signaling deficiency,inhibiting STAT3,or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells,resulting in impaired longevity and antitumor ability. Similarly,human Tc9 cells also exhibited lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8+ T cells expressed lower IL9 and higher lipid peroxidation- and ferroptosis-related genes than circulating CD8+ T cells in patients with melanoma. This study indicates that lipid peroxidation regulates Tc9 cell longevity and antitumor effects via the IL-9/STAT3/fatty acid oxidation pathway and regulating T cell lipid peroxidation can be used to enhance T cell-based immunotherapy in human cancer. View Publication

BACKGROUND Acute respiratory distress syndrome (ARDS) is a neutrophil-associated disease. Delayed neutrophil apoptosis and increased levels of neutrophil extracellular traps (NETs) have been described in ARDS. We aimed to investigate the relationship between these phenomena and their potential as inflammation drivers. We hypothesized that delayed neutrophil apoptosis might enhance NET formation in ARDS. METHOD Our research was carried out in three aspects: clinical research,animal experiments,and in vitro experiments. First,we compared the difference between neutrophil apoptosis and NET levels in healthy controls and patients with ARDS and analyzed the correlation between neutrophil apoptosis and NET levels in ARDS. Then,we conducted animal experiments to verify the effect of neutrophil apoptosis on NET formation in Lipopolysaccharide-induced acute lung injury (LPS-ALI) mice. Furthermore,this study explored the relationship between neutrophil apoptosis and NETs at the cellular level. Apoptosis was assessed using morphological analysis,flow cytometry,and western blotting. NET formation was determined using immunofluorescence,PicoGreen assay,SYTOX Green staining,and western blotting. RESULTS ARDS neutrophils lived longer because of delayed apoptosis,and the cyclin-dependent kinase inhibitor,AT7519,reversed this phenomenon both in ARDS neutrophils and neutrophils in bronchoalveolar lavage fluid (BALF) of LPS-ALI mice. Neutrophils in a medium containing pro-survival factors (LPS or GM-CSF) form more NETs,which can also be reversed by AT7519. Tissue damage can be reduced by promoting neutrophil apoptosis. CONCLUSIONS Neutrophils with extended lifespan in ARDS usually enhance NET formation,which aggravates inflammation. Enhancing neutrophil apoptosis in ARDS can reduce the formation of NETs,inhibit inflammation,and consequently alleviate ARDS. View Publication