搜索结果: 'gmp t cell activator'

In life sciences,the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently,cell rheological measurements were either limited by acquisition throughput,excessive post processing,or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes,but has been restricted to single material parameters as the Young's modulus. Here,we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition,our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties. View Publication

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens,but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells,we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs,40{\%} of antigen-specific SED B cells bind antigen within 2 h,whereas unspecifc cells do not,indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice,but is unperturbed in mice depleted of classical dendritic cells (DC). Thus,we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses,and should be taken into account when developing mucosal vaccines. View Publication

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca-1) cells to reconstitute aged BM and rejuvenate the aged heart,and examined the underlying molecular mechanisms. BM Sca-1+ or Sca-1- cells from young (2-3 months) or aged (18-19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca-1+,young Sca-1-,old Sca-1+,and old Sca-1- . Four months later,expression of rejuvenation-related genes (Bmi1,Cbx8,PNUTS,Sirt1,Sirt2,Sirt6) and proteins (CDK2,CDK4) was increased along with telomerase activity and telomerase-related protein (DNA-PKcs,TRF-2) expression,whereas expression of senescence-related genes (p16INK4a,P19ARF,p27Kip1 ) and proteins (p16INK4a,p27Kip1 ) was decreased in Sca-1+ chimeric hearts,especially in the young group. Host cardiac endothelial cells (GFP- CD31+ ) but not cardiomyocytes were the primary cell type rejuvenated by young Sca-1+ cells as shown by improved proliferation,migration,and tubular formation abilities. C-X-C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP+ ) cells isolated from young Sca-1+ chimeric hearts. Protein expression of Cxcr4,phospho-Akt,and phospho-FoxO3a in endothelial cells derived from the aged chimeric heart was increased,especially in the young Sca-1+ group. Reconstitution of aged BM with young Sca-1+ cells resulted in effective homing of functional stem cells in the aged heart. These young,regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells. View Publication

The complex process of platelet formation originates with the hematopoietic stem cell,which differentiates through the myeloid lineage,matures,and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRalpha+ perivascular cells,derived from human umbilical cord tissue,which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression,yielding platelets that are functional with low basal activation levels,a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo. View Publication

This study aimed to determine the association between non-high-fat diet-induced obesity- (non-DIO-) associated gut microbiome dysbiosis with gut abnormalities like cellular turnover of intestinal cells,tight junctions,and mucin formation that can impact gut permeability. We used leptin-deficient (Lepob/ob) mice in comparison to C57BL/6J control mice,which are fed on identical diets,and performed comparative and correlative analyses of gut microbiome composition,gut permeability,intestinal structural changes,tight junction-mucin formation,cellular turnover,and stemness genes. We found that obesity impacted cellular turnover of the intestine with increased cell death and cell survival/proliferation gene expression with enhanced stemness,which are associated with increased intestinal permeability,changes in villi/crypt length,and decreased expression of tight junctions and mucus synthesis genes along with dysbiotic gut microbiome signature. Obesity-induced gut microbiome dysbiosis is also associated with abnormal intestinal organoid formation characterized with decreased budding and higher stemness. Results suggest that non-DIO-associated gut microbiome dysbiosis is associated with changes in the intestinal cell death versus cell proliferation homeostasis and functions to control tight junctions and mucous synthesis-regulating gut permeability. View Publication

A large body of evidence suggests that B-cell lymphomas with enhanced Myc expression are associated with an aggressive phenotype and poor prognosis,which makes Myc a compelling therapeutic target. Phosphodiesterase 4B (PDE4B),a main hydrolyzer of cyclic AMP (cAMP) in B cells,was shown to be involved in cell survival and drug resistance in diffuse large B cell lymphomas (DLBCL). However,the interrelationship between Myc and PDE4B remains unclear. Here,we first demonstrate the presence of the Myc-PDE4B feed-forward loop,in which Myc and PDE4B mutually reinforce the expression of each other. Next,the combined targeting of Myc and PDE4 synergistically prevented the proliferation and survival of B lymphoma cells in vitro and in a mouse xenograft model. We finally recapitulated this combinatorial effect in Emu-myc transgenic mice; co-inhibition of Myc and PDE4 suppressed lymphomagenesis and restored B cell development to the wild type level that was associated with marked reduction in Myc levels,unveiling the critical role of the Myc-PDE4B amplification loop in the regulation of Myc expression and the pathogenesis of B cell lymphoma. These findings suggest that the disruption of the Myc-PDE4B circuitry can be exploited in the treatment of B cell malignancies. View Publication

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases,but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here,we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines,were characterized by a metabolism typical of quiescent or memory T cells,and did not participate in inflammatory processes. In contrast,infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines,disseminated widely into the periphery,and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population. View Publication

NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases,their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-gamma and TNF-alpha; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article,we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia,which was accompanied by significant reduction in IFN-gamma production by immune cells in the spleens and liver of infected mice. Strikingly,infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-gamma and TNF-alpha) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively,these studies show that NK cells are critical for optimal resistance to T. congolense,and its deficiency leads to enhanced susceptibility in infected mice. View Publication

Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non-barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis,tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non-hematopoietic cells. Here we provide protocols designed for processing the lung,liver,small intestine,bone marrow,mediastinal and mesenteric lymph nodes in order to obtain a purified leukocyte fraction of cells,in which ILC2 enrichment is optimized. In addition,we describe in detail the methodologies used to activate ILC2s and the assays necessary for the detection of their effector cytokines. We highlight the differences in ILC2 characterization within distinct tissues that we have recently identified. {\textcopyright} 2019 by John Wiley Sons,Inc. View Publication

Lupus glomerulonephritis (GN) is an autoimmune disease characterized by immune complex-deposition,complement activation and glomerular inflammation. In lupus-prone NZM2328 mice,the occurrence of lupus GN was accompanied by a decrease in Treg cells and an increase in proinflammatory cytokine-producing T cells. Because IL-33 in addition to IL-2 has been shown to be important for Treg cell proliferation and ST2 (IL-33 receptor) positive Treg cells are more potent in suppressor activity,a hybrid cytokine with active domains of IL-2 and IL-33 was generated to target the ST2+ Treg cells as a therapeutic agent to treat lupus GN. Three mouse models were used: spontaneous and Ad-IFNalpha- accelerated lupus GN in NZM2328 and the lymphoproliferative autoimmune GN in MRL/lpr mice. Daily injections of IL233 for 5 days prevented Ad-IFNalpha-induced lupus GN and induced remission of spontaneous lupus GN. The remission was permanent in that no relapses were detected. The remission was accompanied by persistent elevation of Treg cells in the renal lymph nodes. IL233 is more potent than IL-2 and IL-33 either singly or in combination in the treatment of lupus GN. The results of this study support the thesis that IL233 should be considered as a novel agent for treating lupus GN. View Publication

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta),epidermal growth factor (EGF),insulin-like growth factor 1,bovine fibroblast growth factor (bFGF),and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely,addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF,EGF,and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior,suggesting caution in the interpretation of experiments on cellular activity related to Matrigel,collagen type IV,and possibly other extracellular matrix components. View Publication

To achieve a functional cure for HIV,treatment regimens that eradicate latently HIV-infected cells must be established. For this,many groups have attempted to reactivate latently-infected cells to induce cytopathic effects and/or elicit cytotoxic T lymphocyte (CTL)/NK cell-mediated immune responses to kill these cells. We believe that not only the reactivation of latently-infected cells,but also the induction of strong CTL responses,would be required for this. Here,we used typical immune activators that target pattern recognition receptors (PRRs). For our experimental model,we identified eight SIV-infected cynomolgus monkeys that became natural controllers of viremia. Although plasma viral loads were undetectable,we could measure SIV-DNA by qPCR in peripheral blood mononuclear cells (PBMCs). Using these PBMCs,we screened 10 distinct PRR ligands to measure IFN-alpha and IFN-gamma production. Among these,STING ligands,cGAMP and c-di-AMP,and the TLR7/8 agonist R848 markedly increased cytokine levels. Both R848 and STING ligands could reactivate latently-infected cells in both cynomolgus monkeys and human PBMCs in vitro. Furthermore,c-di-AMP increased the frequency of SIV Gag-specific CD8+ T cells including polyfunctional CD8+ T cells,as compared to that in untreated control or R848-treated cells. Together,STING ligands might be candidates for HIV treatment. View Publication