搜索结果: 'gmp t cell activator'

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here,we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion,RNA and ATAC sequencing on baseline and exhausted CART cells,and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further,IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely,IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore,we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy. The application and therapeutic success of CAR-T cell approaches are limited by the development of T cell exhaustion. Here,Stewart et al discover a role for IL-4 in driving CD8+ CAR-T cell exhaustion and demonstrate the improvement of CAR-T cell effectivity with interruption of IL-4 signalling. View Publication

Inflammatory bowel disease (IBD),encompassing Crohn’s disease (CD) and ulcerative colitis (UC),is a chronic inflammatory condition of the gut affecting both adults and children. Neutrophil extracellular traps (NETs) are structures released by activated neutrophils,potentially contributing to tissue damage in various diseases. This study aimed to explore the presence and role of NETs in pediatric IBD. We compared intestinal biopsies and peripheral blood from 20 pediatric IBD patients (UC and CD) to controls. Biopsy staining and techniques for neutrophil activation were used to assess neutrophil infiltration and NET formation. We also measured the enzymatic activity of key NET proteins and evaluated NET formation in UC patients in remission. Both UC and CD biopsies showed significantly higher levels of neutrophils and NETs compared to controls (p < 0.01),with UC exhibiting the strongest association. Peripheral blood neutrophils from UC patients at diagnosis displayed increased NET formation compared to controls and CD patients. Interestingly,NET formation normalized in UC patients following remission-inducing treatment. This pilot study suggests a potential role for NETs in pediatric IBD,particularly UC. These findings warrant further investigation into the mechanisms of NET involvement and the potential for targeting NET formation as a therapeutic strategy. View Publication

SummaryHere,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.For complete details on the use and execution of this protocol,please refer to Foltz et al.1 Graphical abstract Highlights•Protocol for inducing hESCs or iPSCs to form neural crest stem cells (NCSCs)•Steps for differentiating NCSCs into craniofacial cartilage organoids•Instructions for preparing appropriate media and conditions for differentiation•Guidance for assessing changes in cell and organoid morphology during differentiation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling. View Publication

IntroductionThe role of miRNAs in regulating variable molecular functions has been sought by scientists for its promising utility in regulating the immune response and,hence,in treating various diseases. In hepatocellular carcinoma (HCC) specifically,a reduction in the number and efficiency of circulating and intrahepatic natural killer (NK) cells has been reported. Our project aims to investigate the role of miR-216a-3p in the regulation of NK cell cytotoxicity,especially since it plays a tumor suppressor role in the context of HCC.MethodsTo achieve our aim,we isolated NK cells from the whole blood of 86 patients with HCC and 23 healthy controls. We assessed the expression profile of miR-216a-3p in NK cells of patients and controls. Furthermore,we induced the expression of miR-216a-3p in NK cells isolated from healthy controls,followed by measuring the release of interferon-gamma (IFN-γ),tumor necrosis factor-alpha (TNF-α),perforins (PRF) and granzyme B (GrB) using ELISA as well as NK cells cytolytic activity against Huh7 cells using lactate dehydrogenase (LDH) cytotoxicity assay. After that,we performed an in silico analysis to understand the mechanistic regulation imposed by miR-216a-3p on NK cells to study its impact on one of its potential downstream targets.ResultsOur results have indicated that miR-216a-3p has higher expression in NK cells of patients with HCC,and simulating this elevated expression pattern via forcing miR-216a-3p expression in normal NK cells has negatively impacted the release of TNF- α,IFN- γ,GrB,and PRF. Consequently,a decrease in cell cytolysis was observed. Our in silico analysis revealed that the predicted downstream targets of miR-216a-3p are enriched in the FOXO-signaling pathway. Among those targets is FOXO-1,which has been reported to play a role in NK cell maturation. Thus,we evaluated FOXO-1 expression upon mimicking miR-216a-3p in control NK cells that showed significant downregulation of FOXO-1 on both RNA and protein levels.ConclusionIn conclusion,we report miR-216-3p as a negative regulator of NK cell cytotoxicity. View Publication

The persistence of HIV-1 latency reservoirs in CD4+ T cells is a significant obstacle for curing HIV-1. Shock-and-kill strategies,which aim to reactivate latent HIV-1 followed by cytotoxic clearance,have shown limited success in vivo due to insufficient efficacy of latency reversal agents (LRAs) and off-target effects. Natural killer (NK) cells,with their ability to mediate cytotoxicity independent of antigen specificity,offer a promising avenue for enhancing the shock-and-kill approach. Previously,we observed that pan-caspase inhibitors induce NK cells to secrete an LRA in vitro. Here,we aimed to identify this LRA using a targeted proteomic approach. We identified lymphotoxin-α (LTα) as the key LRA secreted by NK cells following pan-caspase inhibitor treatment. LTα was shown to significantly induce HIV-1 LTR promoter activity,a hallmark of viral reactivation. Neutralization of LTα effectively abolished the observed LRA activity,confirming its central role. Moreover,cytokine-primed but not resting human primary NK cells exhibited LRA activity that could be neutralized with LTα neutralizing antibodies. Finally,pan-caspase inhibitor treatment did not decrease the ability of the cytokine-primed NK cells to kill target cells. These findings demonstrate that cytokine-primed NK cells,through LTα secretion,can effectively reactivate latent HIV-1 following pan-caspase inhibitor treatment,without compromising NK cell cytotoxicity. This highlights a potential enhancement strategy utilizing NK cells for shock-and-kill approaches in HIV-1 cure research. View Publication

AbstractObjectivesWe performed multiparameter phenotyping of peripheral B cells in anti-Jo-1 antibody positive idiopathic inflammatory myopathies (IIM) to delineate disease-associated immunological profiles and the influence of B cells on disease activity.MethodsPurified B cells from peripheral blood mononuclear cells from 16 patients with anti-Jo-1 antibody positive IIM (7 with untreated active IIM,4 with active and treated IIM and 5 with inactive IIM) were analysed by multiparameter spectral flow cytometry. Dimensionality reduction and clustering analysis were applied to pre-gated CD19+B cells. Serum levels of 21 cytokines and anti-Jo-1 IgG autoantibodies were determined. All patients with IIM in this study were positive for anti-Jo-1 antibody.ResultsAnti-Jo-1 antibody levels correlated positively to disease activity. Flow cytometry demonstrated B-cell dysregulation with significantly lower CD73 expression on naïve,switched memory and double negative B cells in patients with active IIM. Clustering analysis further revealed expansions of CD73− IgM+naïve B cells and CD73− CD95+ switched memory B cells in active IIM. In unswitched memory B cells,CD73+CD21+ cells were decreased in active IIM. Patients with active IIM had significantly higher serum levels of B-cell activating factor,inducible protein-10,interleukin-6 and sCD40L which correlated with changes in B-cell populations.ConclusionsSince CD73 has an immunoregulatory function by modulating the ATP/adenosine pathway,which is also targeted by methotrexate,the low CD73 B-cell expression in anti-Jo-1 antibody-positive IIM may lead to B-cell hyperactivation. These novel findings further highlight B cells as central in the pathogenesis of IIM and important therapeutic targets. View Publication

The prognosis for adult B-cell acute lymphoblastic leukemia remains unfavorable,especially in the context of relapsed and refractory disease. Exploring the molecular mechanisms underlying disease progression holds significant promise for improving clinical outcomes. In this investigation,utilizing single-cell transcriptome sequencing technology,we discerned a correlation between Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) and the progression of B-cell acute lymphoblastic leukemia. Our findings reveal a significant upregulation of UHRF1 in cases of relapsed and refractory B-cell acute lymphoblastic leukemia,thereby serving as a prognostic indicator for poor outcomes. Both deletion of UHRF1 or overexpression of its downstream target secreted frizzled-related protein 5 (SFRP5) resulted in the inhibition of leukemia cell proliferation,promoting cellular apoptosis and induction of cell cycle arrest. Our results showed that UHRF1 employs methylation modifications to repress the expression of SFRP5,consequently inducing the WNT5A-P38 MAPK-HK2 signaling axis,resulting in the augmentation of lactate,the critical metabolic product of aerobic glycolysis. Furthermore,we identified UM164 as a targeted inhibitor of UHRF1 that substantially inhibits P38 protein phosphorylation,downregulates HK2 expression,and reduces lactate production. UM164 also demonstrated antileukemic activity both in vitro and in vivo. In summary,our investigation revealed the molecular mechanisms of epigenetic and metabolic reprogramming in relapsed and refractory B-cell acute lymphoblastic leukemia and provides potential targeted therapeutic strategies to improve its inadequate prognosis. View Publication

SummaryStudying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression,such as experimental autoimmune encephalomyelitis (EAE),is often limited by the availability of gene-edited mice. Here,we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction,preparation of retroviruses,viral delivery,and Th17 differentiation. We then detail procedures for in vivo functionality analysis.For complete details on the use and execution of this protocol,please refer to Zhong et al.1,2 Graphical abstract Highlights•Steps for designing and cloning dual U6gRNA cassettes to delete a specific CRE•Instructions for optimized retrovirus production and transduction into CD4+ T cells•Guidance on Th17 differentiation and confirmation of CRE deletion in cultured T cells•Procedures for adoptive transfer of CRISPR-edited Th17 cells to assess in vivo function Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression,such as experimental autoimmune encephalomyelitis (EAE),is often limited by the availability of gene-edited mice. Here,we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction,preparation of retroviruses,viral delivery,and Th17 differentiation. We then detail procedures for in vivo functionality analysis. View Publication

Enhancers and transcription factors (TFs) are crucial in regulating cellular processes. Current multiomic technologies to study these elements in gene regulatory mechanisms lack multiplexing capability and scalability. Here we present single-cell ultra-high-throughput multiplexed sequencing (SUM-seq) for co-assaying chromatin accessibility and gene expression in single nuclei. SUM-seq enables profiling hundreds of samples at the million cell scale and outperforms current high-throughput single-cell methods. We demonstrate the capability of SUM-seq to (1) resolve temporal gene regulation of macrophage M1 and M2 polarization to bridge TF regulatory networks and immune disease genetic variants,(2) define the regulatory landscape of primary T helper cell subsets and (3) dissect the effect of perturbing lineage TFs via arrayed CRISPR screens in spontaneously differentiating human induced pluripotent stem cells. SUM-seq offers a cost-effective,scalable solution for ultra-high-throughput single-cell multiomic sequencing,accelerating the unraveling of complex gene regulatory networks in cell differentiation,responses to perturbations and disease studies. This work presents SUM-seq,an ultra-high-throughput method for co-profiling chromatin accessibility and gene expression in single nuclei across multiplexed samples,advancing the study of gene regulation in diverse biological systems. View Publication

ObjectiveB lymphocytes play a crucial role in immunity but also contribute to the pathogenesis of various diseases. Cannabis plants produce numerous biologically active compounds,including cannabinoids. The two most studied phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These cannabinoids exert diverse and potent biological effects primarily through the endocannabinoid system (ECS),which also plays a key role in mature B-cell function. Both the immune system and the ECS undergo age-related changes that lead to a clinically significant decline in function.MethodsThis study compares the effects of THC and CBD on B-cell activity in young and aged mice. Murine B lymphocytes were activated using lipopolysaccharide (LPS) and interleukin-4 (IL-4),and the impact of cannabinoid treatments was assessed in terms of cell phenotype,proliferation,antibody secretion,tumor necrosis factor-alpha (TNFα) secretion,extracellular signal-regulated kinase (ERK) phosphorylation,and the cellular metabolome.ResultsBoth THC and CBD exhibited dose-dependent inhibitory effects on B-cell activation in young and aged mice. However,we show here,for the first time,that the treatments induce distinct metabolic profiles. Although some metabolites,such as glucose-6-phosphate,pentose phosphate pathway (PPP) and nucleotide metabolites,were reduced by both cannabinoids,THC selectively reduced the levels of a distinct set of amino acids,while only CBD increased the levels of Citrulline and Allantoin. Additionally,the effects of THC and CBD differed between young and aged B cells,suggesting that age-related changes in the ECS may influence cannabinoid sensitivity.ConclusionsThese findings provide insights into the distinct mechanisms by which THC and CBD regulate immune activation and may open the door for investigating the mechanisms behind cannabinoids effects on the immune system. They also highlight the need for further research into phytocannabinoid-based therapies,particularly in age-specific contexts. Given the immunoregulatory properties of cannabinoids,especially CBD,tailored therapeutic strategies may enhance their clinical applications View Publication

The long-term maintenance of hematopoietic stem cells (HSCs) relies on the regulation of endoplasmic reticulum (ER) stress at a low level,but the underlying mechanism remains poorly understood. Here,we demonstrate that suppression of ER stress improves the functions of HSCs and protects HSCs against ionizing radiation (IR)-induced injury. We identify epithelial membrane protein 1 (EMP1) as a key regulator that mitigates ER stress in HSCs. Emp1 deficiency leads to the accumulation of protein aggregates and elevated ER stress,ultimately resulting in impaired HSC maintenance and self-renewal. Mechanistically,EMP1 is located within the ER and interacts with ceramide synthase 2 (CERS2) to limit the production of a class of sphingolipids,dihydroceramides (dhCers). DhCers accumulate in Emp1-deficient HSCs and induce protein aggregation. Furthermore,Emp1 deficiency renders HSCs more susceptible to IR,while overexpression of Emp1 or inhibition of CERS2 protects HSCs against IR-induced injury. These findings highlight the critical role played by the EMP1-CERS2-dhCers axis in constraining ER stress and preserving HSC potential. A new study shows EMP1 protects hematopoietic stem cells by suppressing sphingolipid metabolism and ER stress. EMP1 interacts with CERS2 to limit dihydroceramide production,which causes protein aggregation when elevated. View Publication

BACKGROUND Emerging evidence of antibody-independent functions,as well as the clinical efficacy of anti-CD20 depleting therapies,helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. OBJECTIVE To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB),resembling classical cytotoxic CD8+ T lymphocytes,in the peripheral blood from relapsing-remitting MS (RRMS) patients. METHODS In this study,104 RRMS patients during different treatments and 58 healthy donors were included. CD8,CD19,Runx3,and GzmB expression was assessed by flow cytometry analyses. RESULTS RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA),untreated RRMS patients,and healthy donors but not when compared to interferon-$\beta$ (IFN). Moreover,regarding Runx3,the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes,the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. CONCLUSIONS CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future,monitoring cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions." View Publication