技术资料

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses,there is an exciting prospect of further enhancing CAR functionality. Here,we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4),myeloid differentiation primary response 88 (MyD88),or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-β (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model,MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors. View Publication

Although platinum-based chemotherapy is the frontline regimen for colorectal cancer (CRC),drug resistance remains a major challenge affecting its therapeutic efficiency. However,there is limited research on the correlation between chemotherapy resistance and lipid metabolism,including PIK3CA mutant tumors. In this present study,we found that PIK3CA-E545K mutation attenuated cell apoptosis and increased the cell viability of CRC with L-OHP treatment in vitro and in vivo . Mechanistically,PIK3CA-E545K mutation promoted the nuclear accumulation of SREBP1,which promoted the transcription of Apolipoprotein A5 (APOA5). APOA5 activated the PPARγ signaling pathway to alleviate reactive oxygen species (ROS) production following L-OHP treatment,which contributed to cell survival of CRC cells. Moreover,APOA5 overexpression enhanced the stemness-related traits of CRC cells. Increased APOA5 expression was associated with PIK3CA mutation in tumor specimens and poor response to first-line chemotherapy,which was an independent detrimental factor for chemotherapy sensitivity in CRC patients. Taken together,this study indicated that PIK3CA-E545K mutation promoted L-OHP resistance by upregulating APOA5 transcription in CRC,which could be a potent target for improving L-OHP chemotherapeutic efficiency. Our study shed light to improve chemotherapy sensitivity through nutrient management in CRC. View Publication

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here,we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19,the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation. View Publication

Precise gene editing uncovers mis-splicing of BUBR1 and CDC27 in human SF3B1-mutant HSPCs,leading to activation of mitotic checkpoint and rendering the cells sensitive to CHK1 inhibitor prexasertib. View Publication

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor; GBM’s inevitable recurrence suggests that glioblastoma stem cells (GSC) allow these tumors to persist. Our previous work showed that FOSL1,transactivated by the STAT3 gene,functions as a tumorigenic gene in glioma pathogenesis and acts as a diagnostic marker and potential drug target in glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. To investigate downstream molecules of FOSL1,we analyzed the transcriptome after overexpressing FOSL1 in a PDX-L14 line characterized by deficient FOSL1 expression. We then conducted immunohistochemical staining for FOSL1 and NF-κB p65 using rabbit polyclonal anti-FOSL1 and NF-κB p65 in glioma tissue microarrays (TMA) derived from 141 glioma patients and 15 healthy individuals. Next,mutants of the human FOSL1 promoter,featuring mutations in essential binding sites for NF-κB were generated using a Q5 site-directed mutagenesis kit. Subsequently,we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. Then,we explored the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently,we assessed the activity of FOSL1 and NF-κB. To understand the role of FOSL1 in cell growth and stemness,we conducted a CCK-8 assay and cell cycle analysis,assessing apoptosis and GSC markers,ALDH1,and CD133 under varying FOSL1 expression conditions. Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathway is regulated by FOSL1. NF-κB p65 protein expression correlates to the expression of FOSL1 in glioma patients,and both are associated with glioma grades. NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibiting apoptosis. Therefore,the FOSL1 molecular pathway is functionally connected to NF-κB activation,enhances stemness,and is indicative that FOSL1 may potentially be a novel GBM drug target. The online version contains supplementary material available at 10.1007/s00018-024-05293-1. View Publication

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here,we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro,both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model,transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease. Subject terms: Targeted gene repair,Sickle cell disease View Publication

Ugale et al. demonstrate that CDC42,ERK,and mTORC1 signaling are polarized in premitotic hematopoietic stem cells and unequally segregated during asymmetric cell division. A CDC42/ERK/mTORC1 pathway maintains HSC polarity and balances symmetric and asymmetric cell division. View Publication

Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However,whether and how NETs contribute to sustained airway inflammation in COPD remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD,employing human neutrophils,airway epithelial cells (AECs),dendritic cells (DCs),and a long-term CS-induced COPD mouse model,alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice ( cGAS -−/−,TLR9 −/− ); Additionally,bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to mitochondrial respiratory chain dysfunction. These CSE-NETs,containing oxidatively-damaged DNA (NETs-DNA),promoted AECs proliferation,nuclear factor kappa B (NF-κB) activation,NF-κB-dependent cytokines and type-I interferons production,and DC maturation,which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model,blocking NETs-DNA-sensing via cGAS − /− and TLR9 − /− mice,inhibiting NETosis using mitoTEMPO,and degrading NETs-DNA with DNase-I,respectively,reduced NETs infiltrations,airway inflammation,NF-κB activation and NF-κB-dependent cytokines,but not type-I interferons due to IFN-α/β receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity) in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels,but not type-I interferon levels. In conclusion,NETs-DNA promotes NF-κB-dependent autoimmunity via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore,targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD. Subject terms: Inflammation,Respiratory tract diseases View Publication

Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group,respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1,7,14,28,35,42,49,56,and 63 posttransplantation. BLI signals were detected in all recipients,including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However,the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore,our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts,as confirmed by insulin and glucagon staining. Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy. View Publication

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies,which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells,have driven additional investigations,increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods,we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media,SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold,while absence of Beta-2 microglobulin,a key component of major histocompatibility complex class I molecules,has been reported to reduce the immunogenicity of lentiviral particles. Furthermore,we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion,reduces handling,and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34 + hematopoietic stem cells. View Publication

Mononuclear phagocytes (MNP),including macrophages and dendritic cells form an essential component of primary responses to environmental hazards and toxic exposures. This is particularly important in disease conditions such as asthma and allergic airway disease,where many different cell types are present. In this study,we differentiated CD34+ haematopoietic stem cells towards different populations of MNP in an effort to understand how different cell subtypes present in inflammatory disease microenvironments respond to the common allergen house dust mite (HDM). Using single cell mRNA sequencing,we demonstrate that macrophage subtypes MC SPP1+ and MLC MARCO+ display different patterns of gene expression after HDM challenge,noted especially for the chemokines CXCL5,CXCL8,CCL5 and CCL15. MLC CD206Hi alternatively activated macrophages displayed the greatest changes in expression,while neutrophil and monocyte populations did not respond. Further work investigated how pollutant diesel exhaust particles could modify these transcriptional responses and revealed that CXC but not CC type chemokines were further upregulated. Through the use of diesel particles with adsorbed material removed,we suggest that soluble pollutants on these particles are the active constituents responsible for the modifying effects on HDM. This study highlights that environmental exposures may influence tissue responses dependent on which MNP cell type is present,and that these should be considerations when modelling such events in vitro. Understanding the nuanced responsiveness of different immune cell types to allergen and pollutant exposure also contributes to a better understanding of how these exposures influence the development and exacerbation of human disease. View Publication

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However,the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package,factR2,which offers a comprehensive suite of AS-NMD analysis functions. Using this tool,we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably,this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes. The online version contains supplementary material available at 10.1186/s13059-024-03305-8. View Publication