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

BackgroundChimeric antigen receptor T (CAR-T) cell therapy holds promise for cancer treatment,but its efficacy is often hindered by metabolic constraints in the tumor microenvironment. This study investigates the role of glutamine in enhancing CAR-T cell function against ovarian cancer.MethodsMetabolomic profiling of blood samples from ovarian cancer patients treated with MSLN-CAR-T cells was conducted to identify metabolic changes. In vitro,glutamine pretreatment was applied to CAR-T cells,and their proliferation,CAR expression,tumor lysis,and cytokine production (TNF-α,IFN-γ) were assessed. Mechanistic studies focused on the mTOR-SREBP2 pathway and its effect on HMGCS1 expression,membrane stability and immune synapse formation. In vivo,the antitumor effects and memory phenotype of glutamine-pretreated CAR-T cells were evaluated.ResultsElevated glutamine levels were observed in the blood of ovarian cancer patients who responded to MSLN-CAR-T cell treatment. Glutamine pretreatment enhanced CAR-T cell proliferation,CAR expression,tumor lysis,and cytokine production. Mechanistically,glutamine activated the mTOR-SREBP2 pathway,upregulating HMGCS1 and promoting membrane stability and immune synapse formation. In vivo,glutamine-pretreated CAR-T cells exhibited superior tumor infiltration,sustained antitumor activity,and preserved memory subsets.ConclusionsOur findings highlight glutamine-driven metabolic rewiring via the mTOR-SREBP2-HMGCS1 axis as a strategy to augment CAR-T cell efficacy in ovarian cancer.Trial registrationNCT05372692Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06853-0. View Publication

Neural crest induction begins early during neural plate formation,requiring precise transcriptional control to activate lineage-specific enhancers. Here,we demonstrate that SALL4,a transcription factor associated with syndromes featuring craniofacial anomalies,plays a crucial role in early cranial neural crest (CNCC) specification. Using SALL4-het-KO human iPSCs to model clinical haploinsufficiency,we show that SALL4 directly recruits BAF to CNCC-lineage specific enhancers at the neuroectodermal stage,specifically when neural crest gene expression is induced at the neural plate border. Without functional SALL4,BAF is not loaded at chromatin,leaving CNCC enhancers inaccessible. Consequently,the cells cannot undergo proper CNCC induction and specification due to persistent enhancer repression,despite normal neuroectodermal and neural plate progression. Moreover,by performing SALL4 isoform-specific depletion,we demonstrate that SALL4A is the isoform essential for CNCC induction and specification,and that SALL4B cannot compensate for SALL4A loss in this developmental process. In summary,our findings reveal SALL4 as essential regulator of BAF-dependent enhancer activation during early stages of neural crest development,providing molecular insights into SALL4-associated craniofacial anomalies. View Publication

Background and Objectives: Serum myelin oligodendrocyte glycoprotein (MOG) antibodies are a hallmark of the newly defined neuroinflammatory disease entity MOG antibody–associated disease (MOGAD). Yet,the lack of patient-derived recombinant human MOG (hMOG)–reactive autoantibodies limits investigations into the molecular mechanisms by which these autoantibodies mediate CNS pathology,thereby hindering rational therapeutic approaches. To understand the origins and disease-relevant mechanisms of autoantibodies in MOGAD,we generated and characterized monoclonal anti-hMOG antibodies (MOG-mAbs) from circulating B cells of patients with MOGAD.Methods: We isolated MOG-specific B-cell receptor (BCR) sequences from unique circulating B-cell clones of 6 patients with MOGAD using an antigen selection approach. BCR sequences were expressed as immunoglobulin (Ig)G1 antibodies,and their molecular features,epitope specificity,and binding to MOG isoforms were investigated. The MOG-mAbs’ ability to mediate antibody-dependent cellular phagocytosis (ADCP),natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC),and complement-dependent cytotoxicity (CDC) toward MOG-expressing cells was assessed by live cell-based assays.Results: Of the 15 MOG-mAbs generated,4 revealed evidence of affinity maturation,whereas the remaining 11 were germline encoded. Binding capacities to hMOG varied considerably,with the most frequent putative epitope mapping to a region that includes residue P42. The efficacy of these antibodies in mediating ADCP,ADCC,and CDC of MOG-expressing cells was heterogeneous and associated with their binding characteristics to MOG and its isoforms.Discussion: Taken together,the molecular characteristics and binding patterns of these patient-derived MOG-mAbs reveal a diverse repertoire of MOG-binding autoantibodies with pathogenic capacity in vitro. Consequently,these well-characterized patient autoantibodies offer a foundation for developing in vivo models of MOGAD,serve as tools to standardize diagnostic assays,and guide development of therapeutic strategies targeting either B cells or autoantibodies and their effector functions. View Publication

The immunomodulatory function of the gastric microbiota in cancer is poorly understood,partly due to the stomach’s acidic environment and limited microbial colonization. Here,by analyzing 68 paired human gastric cancer (GC) samples,we identify Ligilactobacillus salivarius as a commensal bacterium depleted in tumors but enriched in immune checkpoint blockade (ICB) responders. Oral administration of L. salivarius enhances anti-PD-1 efficacy in multiple GC mouse models by promoting pro-inflammatory macrophage activation. Mechanistically,bacterial extracellular vesicles (bEVs) derived from L. salivarius deliver 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-BdpM) to tumors,where it activates formyl peptide receptor 1 (FPR1) on macrophages,triggering mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling. Moreover,2,3-BdpM augments the cytotoxic activity of chimeric antigen receptor (CAR)-Claudin18.2+ macrophages in an FPR1-dependent manner. These findings describe a microbial-macrophage axis that enhances GC immunotherapy and highlights the translational potential of orally deliverable microbial adjuvants. Graphical abstract Highlights•Intratumoral reduction of L. salivarius correlates with GC immunotherapy efficacy•bEVs derived from L. salivarius enhance immunotherapy efficacy in GC mouse models•2,3-BdpM in bEV triggers pro-inflammatory macrophage remodeling via FPR1•The cytotoxicity of CAR-Claudin18.2+ macrophages was amplified with 2,3-BdpM alone Yu et al. identify Ligilactobacillus salivarius as a gastric commensal enriched in immunotherapy responders. Oral administration enhances anti-PD-1 efficacy by delivering 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-BdpM) via bacterial extracellular vesicles (bEVs) to activate pro-inflammatory macrophages through formyl peptide receptor 1 (FPR1),revealing a microbial-macrophage axis that potentiates gastric cancer immunotherapy. 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

Widespread uses of nuclear materials increase the risk of accidental or intentional radiation exposure,which can result in acute radiation syndrome (ARS). Hematopoietic ARS (H-ARS) occurs at relatively low doses and is potentially lethal without intervention. While several FDA-approved cytokine-based radiomitigators exist,many require repeated dosing,complicating deployment in mass-casualty scenarios. This study evaluated a novel long-acting,murine-reactive granulocyte–macrophage colony-stimulating factor (LA-GM-CSF; mPDM608) as a prophylactic and mitigative countermeasure for H-ARS. Male and female C57BL/6 mice were exposed to lethal or sublethal total body irradiation (TBI) and treated with LA-GM-CSF using single- or multi-dose regimens administered before or after TBI. Safety,30-day survival,hematologic recovery,bone marrow cellularity,serum GM-CSF pharmacokinetics,endothelial injury markers,and cytokine profiles were assessed using standard hematology,histopathology,ELISA,and multiplex assays. LA-GM-CSF was well tolerated at doses up to 30 mg/kg. Single or limited dosing conferred significant survival benefits compared with vehicle controls,with optimal efficacy observed at lower doses (3 mg/kg). Post-TBI administration as a single dose 24 h after exposure markedly improved survival in both sexes,with stronger hematopoietic recovery in males. LA-GM-CSF accelerated recovery of neutrophils,red blood cells,platelets,hematocrit,and sternal megakaryocytes,prolonged circulating GM-CSF levels,and favorably modulated endothelial injury markers and select cytokines. LA-GM-CSF demonstrates strong potential as a next-generation radiation countermeasure,providing robust survival benefit and hematopoietic recovery with minimal dosing. The results shown here support further development for H-ARS management under the FDA Animal Rule. View Publication

The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However,the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. Here we show how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis,as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver,we demonstrate that Eklf expression is initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies,we find that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population,followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis. View Publication

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2,a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation,is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells,providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor,SD-208,inhibits smad2 activation in hematopoietic progenitors,suppresses TGF-beta-mediated gene activation in BM stromal cells,and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore,SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover,in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS. View Publication

Adipose tissue development is associated with neovascularization,which might be exploited therapeutically. We investigated the neovasculogenesis antigenic profile and kinetics in adipose tissue-derived stromal cells (ADSCs) to understand the potential of ADSCs to generate new vessels. Murine and human visceral adipose tissues were processed with collagenase to obtain ADSCs from the stromal vascular fraction. Freshly isolated murine and human ADSCs featured the expression of early markers of endothelial differentiation [uptake of DiI-labeled acetylated LDL,CD133,CD34,kinase insert domain receptor (KDR)],but not markers for more mature endothelial cells (CD31 and von Willebrand factor). In methylcellulose medium,multilocular cells positive for Oil Red O staining appeared after 6 days. After 10 days,clusters of ADSCs spontaneously formed branched tubelike structures,which were strongly positive for CD34 and CD31,while losing their ability to undergo adipocyte differentiation. In Matrigel,in the presence of endothelial growth factors ADSCs formed branched tubelike structures. By clonal assays in methylcellulose we also determined the frequency of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming units from ADSCs,compared with bone marrow-derived stromal cells (BMSCs) used as a positive control. After 4-14 days,BMSCs formed 8 +/- 3 BFU-E and 40 +/- 10 CFU-GM,while ADSCs never produced colonies of myeloid progenitors. The developing adipose tissue has neovasculogenic potential,based on the recruitment of local rather than circulating progenitors. Adipose tissue might therefore be a viable autonomous source of cells for postnatal neovascularization. View Publication

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast,early hematopoietic,and vascular development. We have previously shown that an upstream enhancer,Eng -8,together with the promoter region,mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements,we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8,+7+9 enhancers in both blood and endothelial cells. By contrast Pu.1,an Ets factor specific to the blood lineage,and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence. View Publication

Hematopoietic stem cells (HSCs) have the capacity to self-renew and continuously differentiate into all blood cell lineages throughout life. At each branching point during differentiation,interactions with the environment are key in the generation of daughter cells with distinct fates. Here,we examined the role of the cell adhesion molecule JAM-C,a protein known to mediate cellular polarity during spermatogenesis,in hematopoiesis. We show that murine JAM-C is highly expressed on HSCs in the bone marrow (BM). Expression correlates with self-renewal,the highest being on long-term repopulating HSCs,and decreases with differentiation,which is maintained longest among myeloid committed progenitors. Inclusion of JAM-C as a sole marker on lineage-negative BM cells yields HSC enrichments and long-term multilineage reconstitution when transferred to lethally irradiated mice. Analysis of Jam-C-deficient mice showed that two-thirds die within 48 hours after birth. In the surviving animals,loss of Jam-C leads to an increase in myeloid progenitors and granulocytes in the BM. Stem cells and myeloid cells from fetal liver are normal in number and homing to the BM. These results provide evidence that JAM-C defines HSCs in the BM and that JAM-C plays a role in controlling myeloid progenitor generation in the BM. View Publication