搜索结果: 'gmp t cell activator'

AbstractBackgroundHypoxia is associated with the evasion of triple-negative breast cancer (TNBC) from immune surveillance. Hypoxia increases the subpopulation of putative TNBC stem-like cells (TNBCSCs) through activating Wnt/β-Catenin signaling. The shedding of MHC class I-related chain A (MICA) is particularly noteworthy in cancer stem cells (CSCs),promoting the resistance of CSCs to natural killer (NK) cell cytotoxicity. To reestablish MICA/NKG2D-mediated immunosurveillance,we proposed the design of a fusion protein (SHH002-hu1-MICA) which consists of Frizzled-7 (Fzd7)-targeting antibody and MICA,serving as an engager retargeting NK cells against TNBCs,especially TNBCSCs.MethodsOpal multicolor immunohistochemistry staining was used to validate the expression of membrane MICA (mMICA) and existence of NK cells in TNBC tumors; flow cytometry (FCM) assay was used to detect the expression of Fzd7/mMICA on TNBCs. Biolayer interferometry (BLI) and surface plasmon resonance (SPR) assays were executed to assess the affinity of SHH002-hu1-MICA towards rhFzd7/rhNKG2D; near-infrared imaging assay was used to evaluate the targeting capability. A cytotoxicity assay was conducted to assess the effects of SHH002-hu1-MICA on NK cell-mediated killing of TNBCs,and FCM assay to analyze the effects of SHH002-hu1-MICA on the degranulation of NK cells. Finally,TNBC cell-line-derived xenografts were established to evaluate the anti-tumor activities of SHH002-hu1-MICA in vivo.ResultsThe expression of mMICA is significantly downregulated in hypoxic TNBCs and TNBCSCs,leading to the evasion of immune surveillance exerted by NK cells. The expression of Fzd7 is significantly upregulated in TNBCSCs and exhibits a negative correlation with the expression of mMICA and infiltration level of NK cells. On accurate assembly,SHH002-hu1-MICA shows a strong affinity for rhFzd7/rhNKG2D,specifically targets TNBC tumor tissues,and disrupts Wnt/β-Catenin signaling. SHH002-hu1-MICA significantly enhances the cytotoxicity of NK cells against hypoxic TNBCs and TNBCSCs by inducing the degranulation of NK cells and promotes the infiltration of NK cells in CD44high regions within TNBC xenograft tumors,exhibiting superior anti-tumor activities than SHH002-hu1.ConclusionsSHH002-hu1-MICA maintains the targeting property of SHH002-hu1,successfully activates and retargets NK cells against TNBCs,especially TNBCSCs,exhibiting superior antitumor activities than SHH002-hu1. SHH002-hu1-MICA represents a promising new engager for NK cell-based immunotherapy for TNBC. View Publication

AbstractBackgroundChimeric antigen receptor (CAR) T-cell therapy depends on T cells that are genetically modified to recognize and attack cancer cells. Their effectiveness thus hinges on the functionality of a patient’s own T cells. Since CAR T-cell therapy is currently only approved for advanced cancers after at least one line of chemotherapy,we evaluated the potential negative effects of prior exposure to chemotherapy on T-cell functionality.MethodsWe studied T cells of two B-cell non-Hodgkin’s lymphoma patient cohorts,one collected before treatment (pre-therapy) and the other after one or more (median 3) lines of chemotherapy (post-therapy). Leveraging advanced multiparameter flow cytometry,single-cell RNA sequencing (scRNA-seq),whole-genome DNA methylation arrays and in vitro functionality testing of generated CAR T cells,we compared patient samples in their suitability for effective CAR T-cell therapy.ResultsWe discovered significant modifications in T-cell subsets and their transcriptional profiles secondary to chemotherapy exposure. Our analysis revealed a discernible shift towards phenotypically more differentiated T cells and an upregulation of markers indicative of T-cell exhaustion. Additionally,scRNA-seq and DNA methylation analyses revealed gene expression and epigenetic changes associated with diminished functionality in post-therapy T cells. Cytotoxicity assays demonstrated superior killing efficacy of CAR T cells derived from treatment-naïve patients compared with those with chemotherapy history.ConclusionsThese findings corroborate that employing T cells collected prior to frontline chemotherapy could enhance the effectiveness of CAR T-cell therapy and improve patient outcomes. View Publication

Extracellular DNA (eDNA) is crucial for the structural integrity of bacterial biofilms as they undergo transformation from B-DNA to Z-DNA as the biofilm matures. This transition to Z-DNA increases biofilm rigidity and prevents binding by canonical B-DNA-binding proteins,including nucleases. One of the primary defenses against bacterial infections are Neutrophil Extracellular Traps (NETs),wherein neutrophils release their own eDNA to trap and kill bacteria. Here we show that H-NS,a bacterial nucleoid associated protein (NAP) that is also released during biofilm development,is able to incapacitate NETs. Indeed,when exposed to human derived neutrophils,H-NS prevented the formation of NETs and lead to NET eDNA retraction in previously formed NETs. NETs that were exposed to H-NS also lost their ability to kill free-living bacteria which made H-NS an attractive therapeutic candidate for the control of NET-related human diseases. A model of H-NS release from biofilms and NET incapacitation is discussed. View Publication

AbstractBackgroundNatural killer (NK) cells’ unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However,NK cells have demonstrated only moderate responses against cancer in clinical trials.MethodsAdvanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here,we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.ResultsWe observed highly efficient single and multiplex base editing,resulting in significantly enhanced NK cell function in vitro and in vivo. Next,we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt’s lymphoma both in vitro and in vivo.ConclusionsThe use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated. View Publication

Variants of phospholipase C gamma 2 (PLCG2),a key microglial immune signaling protein,are genetically linked to Alzheimer's disease (AD) risk. Understanding how PLCG2 variants alter microglial function is critical for identifying mechanisms that drive neurodegeneration or resiliency in AD. Induced pluripotent stem cell (iPSC) –derived microglia carrying the protective PLCG2 P522R or risk‐conferring PLCG2 M28L variants,or loss of PLCG2,were generated to ascertain the impact on microglial transcriptome and function. Protective PLCG2 P522R microglia showed significant transcriptomic similarity to isogenic controls. In contrast,risk‐conferring PLCG2 M28L microglia shared similarities with PLCG2 KO microglia,with functionally reduced TREM2 expression,blunted inflammatory responses,and increased proliferation and cell death. Uniquely,PLCG2 P522R microglia showed elevated cytokine secretion after lipopolysaccharide (LPS) stimulation and were protected from apoptosis. These findings demonstrate that PLCG2 variants drive distinct microglia transcriptomes that influence microglial functional responses that could contribute to AD risk and protection. Targeting PLCG2‐mediated signaling may represent a powerful therapeutic strategy to modulate neuroinflammation. The impact of Alzheimer's disease protective‐ and risk‐associated variants of phospholipase C gamma 2 (PLCG2) on the transcriptome and function of induced pluripotent stem cell (iPSC) –derived microglia was investigated. PLCG2 risk variant microglia exhibited a basal transcriptional profile similar to PLCG2‐deficient microglia but significantly different from isotype control and the transcriptionally similar PLCG2 protective variant microglia. PLCG2 risk variant and PLCG2‐deficient microglia show decreased levels of triggering receptor expressed on myeloid cells 2 (TREM2). The differential transcriptional pathways of protective and risk‐associated PLCG2 variant microglia functionally affect proliferation,apoptosis,and immune response. Protective PLCG2 microglia show resilience to apoptosis and increased cytokine/chemokine secretion upon exposure to lipopolysaccharide (LPS). View Publication

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis and limited therapeutic options. Leukemic stem cells (LSCs),which drive disease progression and confer resistance to therapy,pose a significant challenge to conventional treatment strategies. In this study,we identified and characterized the inhibitory mechanisms of TH37,a small molecule derived from traditional Chinese medicine,which selectively targets AML blasts and LSCs. Our analyses identified peroxiredoxin 1 (PRDX1),an enzyme that catalyzes the breakdown of hydrogen peroxide (a reactive oxygen species),as the primary molecular target of TH37. We demonstrated that TH37 directly interacts with PRDX1,inhibiting its enzymatic activity and thereby elevating intracellular reactive oxygen species levels in AML cells. PRDX1 was found to be overexpressed in AML,and its expression correlated with poor prognosis and the activation of AML- and cancer-associated pathways. Targeting PRDX1,either through lentiviral short-hairpin RNA-mediated silencing or TH37 treatment,induced apoptosis,reduced colony formation,and impaired the engraftment and growth of AML cells in immunodeficient mouse models. Furthermore,TH37 synergized with conventional chemotherapeutic agent to significantly reduce the viability and colony-forming capacity of AML cells. These findings demonstrate the critical role of PRDX1 in AML pathogenesis and highlight its potential as a key therapeutic target to improve clinical outcomes for AML patients. View Publication

Primary human endothelial cells represent an essential tool to model endothelial dysfunction and to screen interventions for its treatment. Here,we developed a protocol for the synchronous isolation of primary human saphenous vein endothelial cells (HSaVEC),human internal thoracic artery endothelial cells (HITAEC),and human microvascular endothelial cells (HMVEC) from SV and ITA utilized as conduits during coronary artery bypass graft surgery and from subcutaneous adipose tissue excised while providing an access to the heart. Treatment by collagenase type IV and magnetic separation with anti-CD31-antibody-coated beads ensured relatively high efficiency of the isolation (≈60% for HSaVEC,≈50% for HITAEC,and ≈20% for HMVEC) and high purity (≥99%) of isolated ECs within ≈2 weeks (HSaVEC),≈2–3 weeks (HITAEC),and ≈3–4 weeks (HMVEC). A colorimetric assay of cell viability and proliferation,as well as real-time bioimpedance monitoring using the xCELLigence instrument,demonstrated high proliferative activity in HSaVEC,HITAEC,and HMVEC,whilst the in vitro tube formation assay indicated their angiogenic potential. The isolation of HSaVEC,HITAEC,and HMVEC from patients undergoing coronary artery bypass graft surgery is a promising option to investigate endothelial heterogeneity,to interrogate endothelial responses to various stresses,and to pinpoint the optimal approaches for restoring endothelial homeostasis,thereby reproducing them within the bedside-to-bench-to-bedside concept. View Publication

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder,with no effective treatment currently available. Recently,non-pharmacological therapy,especially gamma frequency stimulation has shown promising therapeutic effects in Alzheimer’s disease (AD) mouse models. Electric field (EF) is a non-invasive biophysical approach for neuronal protection. However,whether EF is beneficial in AD neuropathology remains unknown. In this study,we exposed the P301S tauopathy mouse model to EF at gamma frequency on the head. We demonstrated that EF treatment significantly improved the cognitive impairments in the P301S mice. This was accompanied by reduced tau pathologies,suppressed microglial activation,neuroinflammation and oxidative stress in the tauopathy mouse brain. Moreover,EF treatment induced cell-specific responses in neural cells,with neurons being more susceptible,followed by microglia and oligodendrocytes. EF also had favorable effects on synaptic protein in neurons,inflammatory response and complement signaling in microglia,and myelination in oligodendrocytes. This study provides strong evidence that EF at gamma frequency may have great potential to be a novel therapeutic intervention for P301S by attenuating neuropathology and offering neuroprotection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13195-025-01859-8. View Publication

Emerging evidence suggests brain-resident myeloid cells,including perivascular macrophages and microglia,provide a reservoir for HIV infection in the central nervous system (CNS),and their inflammatory activation is a proposed pathogenic mechanism in HIV-associated neurocognitive disorders (HAND). We investigated whether cannabinoid receptor 2 (CB2),an immunomodulatory receptor expressed in myeloid cells,regulates viral replication and inflammation in HIV-infected macrophages and microglia. Using the synthetic CB2-specific agonist JWH-133,we found that CB2 activation reduced HIV replication in primary human monocyte-derived macrophages (MDMs) and human induced pluripotent stem cell-derived microglia (iMg) at differing doses,corresponding to the basal expression of CNR2,which encodes CB2,and related endocannabinoid transcripts in each cell type. JWH-133 broadly reduced release of cytokines from HIV-infected MDMs but not iMg. RNA-seq revealed that CB2 agonism primarily altered interferon and integrated stress response pathways in MDMs while altering homeostatic pathways,including synapse maintenance and phagocytosis,in iMg. Further analyses in iMg revealed that NLRP3 inflammasome activation,but not priming,was reduced by CB2 activation,which did not inhibit HIV-induced nuclear factor kB activation. This study identifies key differences in CB2 response between myeloid lineage cell types and implicates CB2-specific agonists as promising candidates for the regulation of HIV-associated neuroinflammation. View Publication

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However,the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs,no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process,we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study,we used a sized-based,label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie,>3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining,flow cytometry analysis,and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the “downstream processing” of cell manufacturing workflow,ensuring better quality and safety of transplanted cells. View Publication

Acute myeloid leukaemia (AML) is a haematological malignancy characterised by the accumulation of transformed myeloid progenitors in the bone marrow. Piplartine (PL),also known as piperlongumine,is a pro-oxidant small molecule extracted from peppers that has demonstrated antineoplastic potential in solid tumours and other haematological malignancies. In this work,we explored the potential of PL to treat AML through the use of a combination of cellular and molecular analyses of primary and cultured leukaemia cells in vitro and in vivo. We showed that PL exhibits in vitro cytotoxicity against AML cells,including CD34 + leukaemia-propagating cells,but not healthy haematopoietic progenitors,suggesting anti-leukaemia selectivity. Mechanistically,PL treatment increased reactive oxygen species (ROS) levels and induced ROS-mediated apoptosis in AML cells,which could be prevented by treatment with the antioxidant scavenger N -acetyl-cysteine and the pancaspase inhibitor Z-VAD(OMe)-FMK. PL treatment reduced NFKB1 gene transcription and the level of NF-κB p65 (pS536),which was depleted from the nucleus of AML cells,indicating suppression of NF-κB p65 signalling. Significantly,PL suppressed AML development in a mouse xenograft model,and its combination with current AML treatments (cytarabine,daunorubicin and azacytidine) had synergistic effects,indicating translational therapeutic potential. Taken together,these data position PL as a novel anti-AML candidate drug that can target leukaemia stem/progenitors and is amenable to combinatorial therapeutic strategies. Subject terms: Acute myeloid leukaemia,Cancer stem cells,Pharmacology View Publication

Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here,we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABA A receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover,metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03 YEMK . Overall,cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia. Subject areas: Neuroscience,Cellular neuroscience,Stem cells research View Publication