技术资料

Gene edited human pluripotent stem cells are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive immune responses,but have largely not addressed the innate immune cells,such as neutrophils,that mediate inflammation and rejection processes occurring early after graft transplantation. We identify the adhesion molecule ICAM-1 as a hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In our experiments,we find that ICAM-1 blocking or knockout in human pluripotent stem cell-derived cardiovascular therapies imparts significantly diminished binding of multiple immune cell types. ICAM-1 knockout results in diminished T cell proliferation and activation responses in vitro and in longer in vivo retention/protection of knockout grafts following immune cell encounter in NeoThy humanized mice. We also introduce the ICAM-1 knockout edit into existing first-generation hypoimmune human pluripotent stem cells and prevent immune cell binding. This promising hypoimmune editing strategy has the potential to improve transplantation outcomes for regenerative therapies in the setting of cardiovascular pathologies and several other diseases. Hypoimmune gene editing in human pluripotent stem cells (hPSCs) provides a promising platform for cellular therapies. Here,the authors report that CRISPR mediated deletion of ICAM-1 in hPSC-derived grafts reduces immune cell adhesion,dampens T cell activation,and protects against immune rejection. View Publication

High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics,yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput,low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation,allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities,demonstrating efficient delivery of various cargo,with high rates of transfection,gene knockout via non-homologous end joining,and precise knock-in through homology-directed repair. To showcase its utility in functional genomics,we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells,identifying novel regulators of exhaustion,including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-13532-z. View Publication

BackgroundCD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However,its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here,we investigated the role of CD24a,the murine CD24 gene,in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model.MethodsClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations,including myeloid-derived suppressor cells (MDSCs),natural killer cells,T cells,and macrophages,within tumors,spleens,and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors.ResultsKnocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity,effects that were partially reversed by re‐expression of CD24a. Moreover,in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout,underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore,in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1,CXCL5,and CXCL10,chemokines known to recruit gMDSCs,further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation.ConclusionsOur findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state,thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-025-01165-3. View Publication

T-cell exhaustion contributes to immunotherapy failure in chronic lymphocytic leukemia (CLL). Here,we analyze T cells from CLL patients’ blood,bone marrow,and lymph nodes,as well as from a CLL mouse model,using single-cell RNA sequencing,mass cytometry,and tissue imaging. T cells in CLL lymph nodes show the most distinct profiles,with accumulation of regulatory T cells and CD8+ T cells in various exhaustion states,including precursor (TPEX) and terminally exhausted (TEX) cells. Integration of T-cell receptor sequencing data and use of the predicTCR classifier suggest an enrichment of CLL-reactive T cells in lymph nodes. Interactome studies reveal potential immunotherapy targets,notably galectin-9,a TIM3 ligand. Inhibiting galectin-9 in mice reduces disease progression and TIM3+ T cells. Galectin-9 expression also correlates with worse survival in CLL and other cancers,suggesting its role in immune evasion and potential as a therapeutic target. Multi-omics can be used to characterise tumour and immune cell populations. Here the authors use multi-omics to characterise CLL blood and tissue samples and use prediction models for CLL TCR specificity and implicate interactions between galectin-9 and TIM3 as involved in CLL immune escape and propose galectin-9 as a possible immunotherapy target. View Publication

AbstractObjectivesTumour necrosis factor inhibitors (TNFi) are widely used and effective as treatment for immune-mediated inflammatory diseases (IMIDs). However,TNFi therapy causes a faster waning of antibody responses following vaccination. The underlying cause by which TNFi affect humoral immunity remains to be elucidated. The formation of long-lasting,high-affinity antibodies after vaccination results from germinal centre (GC)-derived,T cell-dependent B-cell responses. Therefore,this study investigated how TNFi affect the formation and maintenance of antigen-specific B- and CD4+ T-cell responses following SARS-CoV-2 mRNA vaccination.MethodsSARS-CoV-2 spike-specific B-cell responses were characterised using spectral flow cytometry. Spike-specific CD4+ T cells were measured using an activation-induced marker assay. 15 patients with inflammatory bowel disease (IBD) treated with TNFi were compared with 9 IBD patients without systemic immunosuppression and 10 healthy controls.ResultsSpike-specific CD4+T-cell frequency and phenotype,including T follicular helper cells,were not affected by TNFi. Total spike-specific B-cell frequencies were reduced in TNFi-treated patients. Deep phenotyping revealed lower IgG+memory B-cell frequencies in TNFi-treated patients 3–6 months after vaccination. These data were confirmed in TNFi-treated rheumatoid arthritis patients. Interestingly,already at day 7 after the second vaccination,TNFi therapy reduced the induction of class-switched CD11c- CD71+activated B cells,which are believed to be GC-derived. Conversely,CD11c+B cells,associated with extrafollicular B-cell responses,were not affected by TNFi therapy.ConclusionsThese data suggest that TNFi therapy affects the differentiation of GC-derived B cells,which may explain its effect on humoral immune responses. View Publication

Induction of ferroptosis,an iron-dependent form of regulated cell death,holds promise as a strategy to overcome tumor resistance to conventional therapies and enhance immunotherapy responses. However,while the susceptibility of tumor cells to ferroptosis is extensively studied,limited data exists on the vulnerability of immune cells to disturbed iron balance and lipid peroxidation. Here,we found that T-cell stimulation rewires iron and redox homeostasis and by increasing levels of reactive oxygen species and labile iron promotes lipid peroxidation and T-cells’ ferroptosis. Upon stimulation,we detected changes in the balance of ferroptosis-suppressive proteins,including decrease of GPX4. Subsequently,we identified GPX4 as a master regulator orchestrating T/CAR-T-cells’ sensitivity to ferroptosis and observed that GPX4 inhibitors impair CAR-T cells’ antitumor functions. Our study demonstrated differential GPX4 expression and diverse susceptibility to ferroptosis between CD4⁺ and CD8⁺ T cells. Among analyzed subsets of naïve,central memory (CM),effector memory (EM),and terminally differentiated effector memory (TEMRA),CD8⁺ EM and CD8⁺ TEMRA cells exhibited the highest sensitivity to ferroptosis. We also showed that ferroptosis limited the anti-tumor efficacy of CAR-T cells,while ferroptosis inhibition improved their therapeutic effect,both in vitro and in vivo. Our findings are not only important to understand vulnerabilities of CAR-T cells but may also hold particular significance for their therapeutic development. In this context,future anticancer therapies should be carefully designed to selectively induce the ferroptosis of tumor cells without impeding cytotoxic cells’ antitumor efficacy. Additionally,we postulate that promoting less differentiated phenotype of CAR-T cells should be exploited therapeutically to create CAR-T products characterized by decreased sensitivity to ferroptosis within tumor microenvironment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04133-w. View Publication

CD8+ T cells are central to targeting and eliminating cancer cells. Their function is critically supported by type 1 conventional dendritic cells (cDC1s),which both prime antigen‐specific CD8+ T cells in tumour‐draining lymph nodes (tdLNs) and sustain primed CD8+ T cells within tumours. Despite their importance,the spatiotemporal organisation of cDC1s within tumours and their diverse functional roles remain poorly understood. Here,we use scRNAseq and unbiased spatial analysis to construct a detailed map of cDC1 states and distribution within immunogenic mouse tumours during CD8+ T‐cell‐mediated rejection. We reveal two distinct cDC1 activation states characterised by differential expression of genes linked to anti‐tumour immunity,including Cxcl9 and Il12b. Strikingly,Il12b‐expressing cDC1s are CCR7+ and enriched at tumour borders,where they closely associate with stem‐like TCF1+ CD8+ T cells. In contrast,CCR7–Cxcl9‐expressing cDC1s are preferentially found within the tumour parenchyma alongside effector CD8+ T cells. Analysis of a published dataset of human tumours similarly reveals a spatial association between CCR7+ cDC1 and stem‐like TCF1+ CD8+ T cells. These findings uncover a highly spatially coordinated interaction between cDC1s and CD8+ T cells within tumours,shedding light on the intricate cellular dynamics that underpin effective anti‐tumour immunity. Using scRNAseq and spatial analysis,we analyse cDC1 states and spatial distribution in tumours during immune‐mediated rejection. We identify two cDC1 activation states,each occupying different regions and associated with distinct CD8+ T cell populations. This reveals the spatial organisation of cDC1 states that may be key to anti‐tumour immunity. View Publication

IntroductionT cells are major components of the immune system. Their activation requires interaction between the T cell receptor and co-stimulatory molecules,crucial during infection,inflammation,and allogeneic rejection. Monomeric CRP (mCRP) is a known modulator of inflammation and particularly the innate immune response,however its interaction with T cells as part of the adaptive immune response remains unclear.MethodsPeripheral blood mononuclear cells (PBMC) and T cells were isolated. Flow cytometric analysis was conducted to evaluate Fcγ receptor CD16 expression on T cells,the binding of CRP to T cells,and its impact on proliferation and apoptosis. T cell activation was assessed after 1,2,3,5 and 7 days by assessing CD69 and CD25 expression,and under various conditions including coculture with monocytes and several inhibitory factors.ResultsT cells express CD16 that binds mCRP in a concentration-dependent manner,and particularly on activated T cells. While mCRP reduces apoptosis and accelerates proliferation in T cells,it does not independently activate them. However,activation of monocytes by mCRP leads to T cell activation,indicating a direct cell to cell interaction during CRP-induced activation. This effect could be alleviated by inhibition of the CD80/CD28 pathway.ConclusionCRP does not activate T Cells directly but via PI3-kinase-dependent activation of monocytes and subsequent CD80/CD28 cell to cell contact. The findings suggest the effects of CRP on T cells depend on their environment and the presence of other proinflammatory agents. View Publication

BackgroundMacrophages play a crucial role in the development of early-onset preeclampsia (EOPE),which may be closely associated with an imbalance in macrophage M1/M2 polarization. Mesenchymal stem cell (MSC)-derived apoptotic vesicles (apoVs) have anti-inflammatory,tissue repair,and immunomodulatory functions. MSC-apoVs may ameliorate EOPE by regulating macrophage polarization,but the underlying mechanisms remain to be clarified.MethodsMacrophage infiltration and M1/M2 polarization were first analyzed in the placentas of PE patients and normal pregancies to identify macrophage alterations in EOPE placentas. MSC-apoVs were extracted and characterized. The effects of MSC-apoVs on macrophage polarization and trophoblasts invasion were validated in vivo and in vitro. miRNA transcriptomic sequencing of MSC-apoVs was conducted to identify key miRNAs involved in macrophage M2 polarization and to investigate upstream and downstream regulation factors,which were further validated in vivo and in vitro.ResultsThe proportion of M2 macrophages was significantly reduced in EOPE placentas. MSC-apoVs carrying high levels of miR-191-5p recruited macrophages,downregulated CDK6 protein expression,stabilized mitochondrial membrane potential (MMP),and promoted M2 polarization of macrophages. This enhanced the invasion of trophoblasts and improved EOPE pregnancy outcomes in mice,including reduced blood pressure,decreased urine protein,and improved embryo quality. Overexpression of miR-191-5p mimics in MSC-apoVs further alleviated EOPE-related symptoms,whereas inhibition of miR-191-5p reduced the therapeutic effect of MSC-apoVs. Further experiments confirmed that M2 macrophages polarized by MSC-apoVs promote trophoblasts invasion by secreting platelet-derived growth factor-AB (PDGF-AB),which binds to platelet-derived growth factor receptor-beta (PDGFR-β) on trophoblasts,directly activating the downstream PI3K-AKT-mTOR signaling pathway,thereby improving EOPE.ConclusionOur findings reveal the crucial role of M2 macrophages in the pathogenesis of EOPE. MSC-apoVs with high miR-191-5p recruit macrophages,downregulate CDK6,stabilize MMP,and promote M2 polarization,increasing PDGF-AB secretion,which enhances trophoblasts invasion and thereby treat EOPE. Therefore,MSC-apoVs therapy may serve as a promising strategy to improve the prognosis of EOPE.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04546-5. View Publication

FoxP3+ regulatory T (Treg) cells restrict excessive immune responses and immunopathology as well as reactivity to self or environmental antigens and thus are crucial for peripheral immune tolerance. The transcription factor Interferon Regulatory Factor 4 (IRF4) controls differentiation and function of T cells. In Treg cells,IRF4 is required for peripheral activation and maturation to effector Treg (eTreg) cells with enhanced suppressive function. However,the mechanisms of Treg cell regulation by IRF4 are not fully understood. Here,we analyze the role of IRF4 in differentiation and maintenance of Treg cells using IRF4-deficient mice and a T cell transfer model,that allows Irf4 inactivation in peripheral T cells. We demonstrate that loss of one Irf4 allele already results in impaired eTreg cell differentiation and decreased Treg cell homeostasis,indicating that IRF4 controls peripheral Treg cell differentiation in a gene dosage dependent mode. Peripheral Irf4 inactivation was also associated with enhanced production of inflammatory but also inhibitory cytokines by Treg cells. ATAC sequencing of Treg cells after mutation of one or both Irf4 alleles revealed regions with altered accessibility in genes involved in Treg cell function. In the FoxP3 gene,Irf4 inactivation resulted in reduced ATAC signals in the promoter region and in the conserved non-coding sequence (CNS) 2,required for stability of FoxP3 expression in peripheral Treg cells in response to TCR stimulation. IRF4-deficient Treg cells also displayed a reduction in open chromatin in several Treg cell specific super enhancers,mainly located in proximity to potential IRF4 binding sites. In conclusion,our results demonstrate that IRF4 controls peripheral Treg cell differentiation and homeostasis in a gene dosage dependent manner. View Publication