技术资料

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless,dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS),taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated,the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition,transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together,our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology,Innate immunity,Neural stem cells View Publication

SOT201 and its murine surrogate mSOT201 are novel cis-acting immunocytokines consisting of a humanized/murinized/,Fc-silenced anti-programmed cell death protein 1 (PD-1) monoclonal antibody (mAb) fused to an attenuated human interleukin (IL)-15 and the IL-15Rα sushi+ domain. Murine mPD1-IL2v is a conjugate of a murinized,Fc silenced anti-PD-1 mAb bearing human IL-2 with abolished IL-2Rα binding. These immunocytokines spatiotemporally reinvigorate PD-1 + CD8 + tumor-infiltrating lymphocytes (TILs) via cis-activation and concomitantly activate the innate immunity via IL-2/15Rβγ signaling. Human peripheral blood mononuclear cell and cell lines were used to evaluate cis/trans activity of SOT201. Anti-PD-1 mAb responsive (MC38,CT26) and resistant (B16F10,CT26 STK11 KO) mouse tumor models were used to determine the anticancer efficacy,and the underlying immune cell activity was analyzed via single-cell RNA sequencing and flow cytometry. The expansion of tumor antigen-specific CD8 + T cells by mSOT201 or mPD1-IL2v and memory CD8 + T-cell generation in vivo was determined by flow cytometry. SOT201 delivers attenuated IL-15 to PD-1 + T cells via cis-presentation,reinvigorates exhausted human T cells and induces higher interferon-γ production than pembrolizumab in vitro. mSOT201 administered as a single dose exhibits strong antitumor efficacy with several complete responses in all tested mouse tumor models. While mPD1-IL2v activates CD8 + T cells with a 50-fold higher potency than mSOT201 in vitro,mSOT201 more effectively reactivates effector exhausted CD8 + T cells (Tex),which demonstrate higher cytotoxicity,lower exhaustion and lower immune checkpoint transcriptional signatures in comparison to mPD1-IL2v in MC38 tumors in vivo. This can be correlated with a higher rate of complete responses in the MC38 tumor model following mSOT201 treatment when compared with mPD1-IL2v. mSOT201 increased the relative number of tumor antigen-specific CD8 + T cells,and unlike mPD1-IL2v stimulated greater expansion of adoptively transferred ovalbumin-primed CD8 + T cells simultaneously limiting the peripheral CD8 + T-cell sink,leading to the development of memory CD8 + T cells in vivo. SOT201 represents a promising therapeutic candidate that preferentially targets PD-1 + TILs,delivering balanced cytokine activity for reviving CD8 + Tex cells in tumors. SOT201 is currently being evaluated in the Phase I clinical study VICTORIA-01 ( NCT06163391 ) in patients with advanced metastatic cancer. View Publication

This study aimed to assess the efficacy of Quality and Quantity media-cultured mononuclear cells (QQ-MNCs) for promoting nerve regeneration in a mouse sciatic nerve transection model. Human peripheral blood mononuclear cells (PB-MNCs) and QQ-MNCs derived from healthy volunteers were used/compared. The left sciatic nerve was surgically transected in 27 mice. After complete nerve transection was confirmed,end-to-end direct epineurial nerve repair was performed using 9–0 nylon. Fibrin glue was applied to the tissue around the injury site to limit diffusion of the study treatment followed by application of 0.5 ml phosphate buffered saline (PBS) or PB-MNCs (2x10 6 cells) or QQ-MNCs (2x10 6 cells) to the injury site. The skin was then closed using 6–0 nylon. Histomorphology,immunohistochemistry,electrophysiologic examination,and functional assessment were evaluated at 12-weeks followed by euthanasia and subsequent harvesting of the left sciatic nerves and the left and right gastrocnemius muscles for examination. QQ-MNCs mice exhibited significant improvement in all histomorphologic parameters (axon fiber diameter,myelin thickness,percentage of nerve density) and immunohistochemistry assays (S100,SOX10,GFAP,neurofilament,IL-1β,VEGF,anti-HNA,TNF-α,vWF) compared to PBS mice (all p < 0.05). QQ-MNCs mice also had a significantly higher Basso Mouse Scale score compared to PBS mice ( p = 0.018). The percentage of nerve density adjacent to the injury site was significantly higher in QQ-MNCs mice than in PB-MNCs mice ( p = 0.049). IL-1β expression was significantly lower in QQ-MNCs mice than in PB-MNCs mice ( p = 0.01). QQ-MNCs mice demonstrated significantly better functional and histomorphologic outcomes of nerve regeneration compared to PB-MNCs mice and PBS mice. View Publication

The CEBPA transcription factor is frequently mutated in acute myeloid leukemia (AML). Mutations in the CEBPA gene,which are typically biallelic,result in the production of a shorter isoform known as p30. Both the canonical 42-kDa isoform (p42) and the AML-associated p30 isoform bind chromatin and activate transcription,but the specific transcriptional programs controlled by each protein and how they are linked to a selective advantage in AML is not well understood. Here,we show that cells expressing the AML-associated p30 have reduced baseline inflammatory gene expression and display altered dynamics of transcriptional induction in response to LPS,consequently impacting cytokine secretion. This confers p30-expressing cells an increased resistance to the adverse effects of prolonged exposure to inflammatory signals. Mechanistically,we show that these differences primarily arise from the differential regulation of AP-1 family proteins. In addition,we find that the impaired function of the AP-1 member ATF4 in p30-expressing cells alters their response to ER stress. Collectively,these findings uncover a link between mutant CEBPA,inflammation and the stress response,potentially revealing a vulnerability in AML. Subject terms: Gene regulation,Acute myeloid leukaemia,Transcriptional regulatory elements,Epigenetics in immune cells View Publication

Osteoporosis diagnoses are increasing in the ageing population,and although some treatments exist,these have several disadvantages,highlighting the need to identify new drug targets. G protein-coupled receptors (GPCRs) are transmembrane proteins whose surface expression and extracellular activation make them desirable drug targets. Our previous studies have identified 144 GPCR genes to be expressed in primary human osteoclasts,which could provide novel drug targets. The development of high-throughput assays to assess osteoclast activity would improve the efficiency at which we could assess the effect of GPCR activation on human bone cells and could be utilised for future compound screening. Here,we assessed the utility of a high-content imaging (HCI) assay that measured cytoplasmic-to-nuclear translocation of the nuclear factor of activated T cells-1 (NFATc1),a transcription factor that is essential for osteoclast differentiation,and resorptive activity. We first demonstrated that the HCI assay detected changes in NFATc1 nuclear translocation in human primary osteoclasts using GIPR as a positive control,and then developed an automated analysis platform to assess NFATc1 in nuclei in an efficient and unbiased manner. We assessed six GPCRs simultaneously and identified four receptors (FFAR2,FFAR4,FPR1 and GPR35) that reduced osteoclast activity. Bone resorption assays and measurements of TRAP activity verified that activation of these GPCRs reduced osteoclast activity,and that receptor-specific antagonists prevented these effects. These studies demonstrate that HCI of NFATc1 can accurately assess osteoclast activity in human cells,reducing observer bias and increasing efficiency of target detection for future osteoclast-targeted osteoporosis therapies. View Publication

Lipid dyshomeostasis and tau pathology are present in frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD). However,the relationship between lipid dyshomeostasis and tau pathology remains unclear. We report that GRAM Domain Containing 1B (GRAMD1B),a nonvesicular cholesterol transporter,is increased in excitatory neurons of human neural organoids (HNOs) with the MAPT R406W mutation. Human FTLD,AD cases,and PS19 tau mice also have increased GRAMD1B expression. We show that overexpression of GRAMD1B increases levels of free cholesterol,lipid droplets,and impairs autophagy flux. Modulating GRAMD1B in iPSC-derived neurons also alters key autophagy-related components such as PI3K,phospho-AKT,and p62,as well as phosphorylated tau,and CDK5R1. Blocking GRAMD1B function decreases free cholesterol and lipid droplets. Knocking down GRAMD1B additionally reduces phosphorylated tau,and CDK5R1 expression. Our findings elucidate the role of GRAMD1B in the nervous system and highlight its relevance to FTLD and AD. Subject terms: Diseases of the nervous system,Ageing View Publication

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex,cerebellum and remaining areas. First,each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second,the cerebral regions were subjected to the decellularization process and their respective characterization using molecular,histological,and ultrastructural techniques. Third,the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth,we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells,as a unidirectional model of differentiation. Finally,in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and,in addition,it possesses a combination of structural and biochemical signals (e.g.,neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro. The online version contains supplementary material available at 10.1038/s41598-025-95656-w. View Publication

Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs,FR-GECO1a and FR-GECO1c,based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm,respectively,display large responses to Ca 2+ in vitro (Δ F / F 0 = 6 for FR-GECO1a,18 for FR-GECO1c),are bright under both one-photon and two-photon illumination,and have high affinities (apparent K d = 29 nM for FR-GECO1a,83 nM for FR-GECO1c) for Ca 2+ . FR-GECOs offer sensitive and fast detection of single action potentials in neurons,and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators. Subject terms: Fluorescent proteins,Optogenetics,Zebrafish,Molecular neuroscience,Calcium signalling View Publication

The lymph node (LN) performs essential roles in immunosurveillance throughout the body. Developing in vitro models of this key tissue is of great importance to enhancing physiological relevance in immunoengineering. The LN consists of stromal populations and immune cells,which are highly organized and bathed in constant interstitial fluid flow (IFF). The stroma,notably the fibroblastic reticular cells (FRCs) and the lymphatic endothelial cells (LECs),play crucial roles in guiding T cell migration and are known to be sensitive to fluid flow. During inflammation,interstitial fluid flow rates drastically increase in the LN. It is unknown how these altered flow rates impact crosstalk and cell behavior in the LN,and most existing in vitro models focus on the interactions between T cells,B cells,and dendritic cells rather than with the stroma. To address this gap,we developed a human engineered model of the LN stroma consisting of FRC-laden hydrogel above a monolayer of LECs in a tissue culture insert with gravity-driven interstitial flow. We found that FRCs had enhanced coverage and proliferation in response to high flow rates,while LECs experienced decreased barrier integrity. We added CD4+ and CD8+ T cells and found that their egress was significantly decreased in the presence of interstitial flow,regardless of magnitude. Interestingly,3.0 μ m/s flow,but not 0.8 μ m/s flow,correlated with enhanced inflammatory cytokine secretion in the LN stroma. Overall,we demonstrate that interstitial flow is an essential consideration in the lymph node for modulating LN stroma morphology,T cell migration,and inflammation. View Publication

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. We previously discovered that heme oxygenase 1 (HO1) is crucial for chemoresistance in AML,but the detailed molecular mechanism of that remains unclear. RNA sequencing was conducted to assess transcriptomic changes in three pairs of AML cells after regulating the expression of HO1. The molecular mechanism by which HO1 induces gilteritinib resistance in FLT3-ITD (FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD)) AML was evaluated by quantitative real-time PCR (qRT-PCR),CCK-8,flow cytometry,and western blotting. FLT3-ITD AML mouse models were established to investigate the effects of HO1 expression on gilteritinib resistance in vivo. In these three pairs of AML cells,we discovered that HO1-mediated drug resistance is connected to the interleukin-4-mediated signaling pathway (specifically STAT6) only in MV4-11 cells with the FLT3-ITD mutation. Further findings revealed that HO1 overexpression confers gilteritinib resistance in FLT3-ITD AML cell lines and primary individual specimens. While suppression of HO1 sensitized FLT3-ITD AML cell lines and primary individual specimens to gilteritinib. Mechanistically,western blotting and flow cytometry confirmed that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens. Moreover,tumor-bearing mice were employed to determine that HO1 overexpression conferred gilteritinib resistance in vivo. Collectively,these studies illustrate that HO1 may act as a successful treatment target for gilteritinib-resistant FLT3-ITD AML patients. The online version contains supplementary material available at 10.1186/s12935-025-03757-3. View Publication

Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and can be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by triple negative breast cancer (TNBC) cells via immunofluorescence imaging of patient samples. Image analysis of Cluster of Differentiation 45RA (CD45RA) expression,a naïve T cell specific protein,revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45+ spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered tumor cell expression of PTPRC,the gene that encodes for CD45. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane. View Publication

Suppression of anticancer immune function is a key driver of tumorigenesis. Identifying molecular pathways that inhibit anticancer immunity is critical for developing novel immunotherapeutics. One such molecule that has recently been identified is the carbohydrate polysialic acid (polySia),whose expression is dramatically upregulated on both cancer cells and immune cells in breast cancer patient tissues. The role of polySia in the anticancer immune response,however,remains incompletely understood. In this study,we profile polySia expression on both healthy primary immune cells and on infiltrating immune cells in the tumour microenvironment (TME). These studies reveal polySia expression on multiple immune cell subsets in patient breast tumors. We find that stimulation of primary T-cells and macrophages in vitro induces a significant upregulation of polySia expression. We subsequently show that polySia is appended to a range of different carrier proteins within these immune cells. Finally,we find that selective removal of polySia can significantly potentiate killing of breast cancer cells by innate immune cells. These studies implicate polySia as a significant negative regulator of anticancer immunity. View Publication