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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

Myocardial infarction (MI) is a major global health concern. Although myeloid cells are crucial for tissue repair in emergency haematopoiesis after MI,excessive myelopoiesis can exacerbate scarring and impair cardiac function. Bone marrow (BM) haematopoietic stem cells (HSCs) have the unique capability to replenish the haematopoietic system,but their role in emergency haematopoiesis after MI has not yet been established. Here we collected human sternal BM samples from over 150 cardiac surgery patients,selecting 49 with preserved cardiac function. We show that MI causes detrimental transcriptional and functional changes in human BM HSCs. Lineage tracing experiments suggest that HSCs are contributors of pro-inflammatory myeloid cells infiltrating cardiac tissue after MI. Therapeutically,enforcing HSC quiescence with the vitamin A metabolite 4-oxo-retinoic acid dampens inflammatory myelopoiesis,thereby modulating tissue remodelling and preserving long-term cardiac function after MI. Rettkowski,Romero-Mulero et al. show that myocardial infarction impacts bone marrow haematopoietic stem cells and leads to inflammatory myelopoiesis,which can be dampened by treatment with 4-oxo-retinoic acid,promoting cardiac recovery. 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

Zhang et al. demonstrate that the expression of a mutated CARMIL2 protein in CD28-deficient mice induces most of the developmental and functional consequences known to result from CD28 costimulation and in turn triggers potent tumor-specific T cell responses resistant to PD-1 and CTLA-4 blockade. Naive T cell activation requires both TCR and CD28 signals. The CARMIL2 cytosolic protein enables CD28-dependent activation of the NF-κB transcription factor via its ability to link CD28 to the CARD11 adaptor protein. Here,we developed mice expressing a mutation named Carmil2QE and mimicking a mutation found in human T cell malignancies. Naive T cells from Carmil2QE mice contained preformed CARMIL2QE-CARD11 complexes in numbers comparable to those assembling in wild-type T cells after CD28 engagement. Such ready-made CARMIL2QE-CARD11 complexes also formed in CD28-deficient mice where they unexpectedly induced most of the functions that normally result from CD28 engagement in a manner that remains antigen-dependent. In turn,tumor-specific T cells expressing Carmil2QE do not require CD28 engagement and thereby escape to both PD-1 and CTLA-4 inhibition. In conclusion,we uncovered the overarching role played by CARMIL2-CARD11 signals among those triggered by CD28 and exploited them to induce potent solid tumor–specific T cell responses in the absence of CD28 ligands and immune checkpoint inhibitors. View Publication

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases,including inclusion body myositis (IBM),Sjögren’s syndrome,and systemic lupus erythematosus. While they have diagnostic relevance for IBM,little is known about the autoreactive B cells that produce these antibodies. To address this,we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase,preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation,this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A,the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases. View Publication

Neutrophils are highly abundant in the oral mucosal tissues,and their balanced activation and clearance are essential for immune homeostasis. Here,we demonstrate that neutrophils infected with the bacterial pathogen Porphyromonas gingivalis (Pg) are captured alive by macrophages in a manner that bypasses all known receptor-ligand interactions involved in the phagocytosis of either live or dead cells. Mechanistically,upon interaction with Pg,or its protease RgpB (gingipains),live neutrophils undergo rapid remodeling of their proteomes,generating neoepitopes. N-terminomics-based proteomic profiling identified multiple RgpB cleavage sites on several azurophilic granule proteins that are translocated to the surface of live neutrophils via low-level degranulation and activate macrophage αMβ2 integrin receptors,thus mediating internalization of non-apoptotic neutrophils within macrophage phagosomes. Macrophages with entrapped live neutrophils exhibit phenotypic and transcriptional reprogramming,consistent with inflammatory outcomes in vitro and in vivo. In contrast to the immunosuppressive outcomes associated with efferocytosis of apoptotic neutrophils,live neutrophil entrapment failed to fully activate several catabolic and metabolic processes and exhibited a defective activation of PPAR-γ mediated pro-resolution pathways,thereby promoting bacterial persistence and hindering the resolution of inflammation. Thus,our data demonstrate a novel immune subversion strategy unique to Pg and reveal a previously unknown mode of live neutrophil sequestration into macrophages during an infection. View Publication

BackgroundAlthough both pre-clinical and clinical studies show promising outcomes,resulting in rapid growth of clinical trials of MSC-based therapies in recent years,the heterogeneity and therapeutic inconsistency of MSCs have severely hampered their clinical applications. Purifying homogenous MSC populations with enhanced specific functions represents one promising approach. We have demonstrated recently that the CD317+ MSCs have enhanced anti-inflammatory functions and improved therapeutic efficacy and consistency.MethodsIn the current study,we performed both in vitro and in vivo investigations to delineate whether and how CD317 regulates the immune modulation function of MSCs.ResultsOur data here indicate that the CD317 directly contributes to the immune suppression function of MSCs stimulated by TNF-α through up-regulating TSG6 via CD317/lipid-raft/TNFR1 complex. The CD317 stabilizes the TNFR1 complex,resulting in hyper-activation of the NF-κB pathway and up-regulation of TSG6,which confers the therapeutic effects of MSCs on the mouse model of ALI (acute lung injury) and IBD (inflammatory bowel disease).ConclusionsThus,the CD317 stabilizes TNFR1 and confers the anti-inflammatory functions of MSCs via NF-κB/TSG6 Pathway.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04527-8. View Publication

SummaryA strong interest in drugs targeting the tumor microenvironment (TME) necessitates new experimental systems that incorporate key TME components. Compared to traditional 2D cell lines,3D ex vivo spheroids from patient-derived xenograft (PDX) materials may better capture patient tumor characteristics. We developed and validated a 3D tumor spheroid model from non-small cell lung cancer (NSCLC) PDXs to enable T cell infiltration. Histologic and transcriptomic analysis suggested that tumor spheroids closely recapitulate the source PDX tumor tissues. Consistent T cell infiltration into tumor spheroids was achieved using a well-established magnetic nanoparticle technology,which maintained T cell function and tumor-killing activity. Drug treatment studies with immunotherapy agents also demonstrated the potential scalability of 3D tumor-T cell spheroids in assessing drug activity,including tumor viability and cytokine secretion. This platform provides a useful tool for evaluating drug candidates that can be translated to patient tumor responses related to both tumor intrinsic and TME factors. Graphical abstract Highlights•We developed a 3D tumor spheroid model from lung cancer patient-derived xenografts•The model enabled robust T cell infiltration and preserved T cell cytotoxic functions•Histology and RNA-seq showed that tumor spheroids closely resembled source tumors•Proof-of-concept experiments showed this platform’s utility in preclinical drug testing Biological sciences; Biotechnology; Natural sciences; Tissue Engineering View Publication

BackgroundT cells are contributors to atherosclerosis pathogenesis. Granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells,a specialized helper T cell subset that highly expresses GM-CSF but lacks other helper T cell markers,could exacerbate atherosclerosis development. Calycosin has been reported to suppress atherosclerosis progression. However,the effect of calycosin on ThGM cells is unknown. This study was designed to test the calycosin-induced impact on the pro-atherosclerotic function of ThGM cells in a mouse atherosclerosis model.MethodsApolipoprotein E knockout (ApoE−/−) mice were fed a high-fat diet and calycosin. The phenotype and cytokine expression of aortic ThGM cells were assessed by flow cytometry. Calycosin-derived influences on ThGM cell differentiation,proliferation,and function were determined by flow cytometry,quantitative RT-PCR,Immunoblotting,gene silencing assays,and co-culture with macrophages.ResultsAortic ThGM cell frequency was attenuated after calycosin administration. Live aortic ThGM cells,phenotypically featuring CD4+CCR6−CCR8−CXCR3−CCR10+,showed slower proliferation and weaker macrophage-activating capability in calycosin-treated mice. Besides,calycosin repressed in vitro ThGM cell differentiation and subsequently impaired ThGM cell-mediated macrophage activation,oxidized low-density lipoprotein (Ox-LDL) uptake,and foam cell formation. Importantly,calycosin upregulated nuclear receptor subfamily 4 group A member 3 (NR4A3) in ThGM cells. NR4A3 silencing partially restored the function of calycosin-treated ThGM cells.ConclusionCalycosin inhibits ThGM cell activity to suppress ThGM-cell-mediated activation of pro-atherosclerotic macrophages to ultimately ameliorate atherosclerosis progression. Therefore,we revealed a novel mechanism by which calycosin protects against atherosclerosis. 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

AbstractBackgroundAdoptive transfer of chimeric antigen receptor (CAR)-expressing natural killer (NK) cells has demonstrated success against hematological malignancies. Efficacy against solid tumors has been limited by poor NK cell survival and function in the suppressive tumor microenvironment (TME). To enhance efficacy against solid tumors,stimulatory cytokines have been incorporated into CAR-NK cell therapeutic approaches. However,current cytokine strategies have limitations,including systemic toxicities,exogenous dependencies,and unwanted TME bystander effects. Here,we aimed to overcome these limitations by modifying CAR-NK cells to express a constitutively active interleukin (IL)-7 receptor,termed C7R,capable of providing intrinsic CAR-NK cell activation that does not rely on or produce exogenous signals nor activate bystander cells.MethodsWe examined persistence,antitumor function,and transcriptional profiles of CAR-NK cells coexpressing C7R in a novel tumor immune microenvironment (TiME) co-culture system and against hematologic and solid tumor xenografts in vivo.ResultsPeripheral blood NK cells expressing a CAR directed against the solid tumor antigen GD2 and modified with C7R demonstrated enhanced tumor killing and persistence in vitro compared with CAR-NK cells without cytokine support and similar functions to CAR-NK cells supplemented with recombinant IL-15. C7R.CAR-NK cells exhibited enhanced survival and proliferation within neuroblastoma TiME xenografts in vivo but produced poor long-term tumor control compared with CAR-NK cells supplemented with IL-15. Similar results were seen using C7R-expressing CD19.CAR-NK cells against CD19+leukemia xenografts. Gene expression analysis revealed that chronic signaling via C7R induced a transcriptional signature consistent with intratumor stressed NK cells with blunted effector function. We identified gene candidates associated with chronic cytokine-stressed NK cells that could be targeted to reduce CAR-NK cell stress within the solid TME.ConclusionC7R promoted CAR-NK cell survival in hostile TMEs independent of exogenous signals but resulted in poor antitumor function in vivo. Our data reveals the detrimental role of continuous IL-7 signaling in CAR-NK cells and provides insights into proper application of cytokine signals when attempting to enhance CAR-NK cell antitumor activity. View Publication