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The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells,whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN,including pre-TFH cells,memory TFH cells,germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages,including multiple responses targeting internal influenza virus proteins,and found that each TFH cell state was attainable within a clonal lineage. Thus,human TFH cells form a durable and dynamic multitissue network. Schattgen et al. profiled the subsets and clonality of CD4+ TFH cells in the blood and lymph nodes of human volunteers who received two influenza vaccines 1 year apart to characterize their dynamics and clonal evolution over 2 years. View Publication

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However,>50% of CD19-CAR-T-treated patients experience progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore,it remains a challenge to efficiently,cost-effectively,and consistently manufacture clinically relevant numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector,Quantum pBac™ (qPB),we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here,we demonstrate in vitro and in vivo that consistent,robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular,we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently,rapidly eradicated tumors,and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore,the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies. View Publication

AbstractIntroductionNovel strategies are needed to address the rising burden of osteoporosis and fragility fractures. High-intensity resistance and impact (HiRIT) exercise has shown benefit in improving bone density in postmenopausal women with osteoporosis/osteopenia. Whether HiRIT can enhance the therapeutic effects of osteoporosis pharmacotherapy has not been established. ROLEX-DUO is a randomised controlled trial designed to assess the efficacy of romosozumab on various bone and muscle outcomes in combination with different exercise interventions in women with postmenopausal osteoporosis/osteopenia.Methods and analysisROLEX-DUO is an 8-month randomised placebo-controlled trial conducted at two tertiary referral centres for patients with osteoporosis/osteopenia in Sydney,New South Wales,Australia. The study is implementing the combination of romosozumab or placebo with different forms of exercise in postmenopausal women with osteoporosis/osteopenia without recent fragility fracture (n=102). Eligible women will be randomised 1:1:1 into one of three groups: (1) romosozumab with supervised HiRIT,(2) romosozumab with unsupervised low-intensity exercise or (3) placebo with unsupervised low-intensity exercise. Co-primary outcomes are the mean percentage change in lumbar spine bone mineral density (BMD),and mean change in five times sit-to-stand test performance (seconds) at 8 months. Secondary/exploratory outcomes include BMD changes at the femoral neck,total hip and distal radius,three-dimensional dual-energy X-ray absorptiometry (DXA) hip outcomes,DXA-derived lean and fat mass,serum markers of bone turnover (procollagen type 1 peptide,C-telopeptide of type 1 collagen) and bone biomarkers (dickkopf-1),serum extracellular vesicle analyses,36-Item Short Form Survey (SF-36) quality-of-life scores,Menopause-Specific Quality Of Life (MENQOL) Questionnaire menopause symptom burden scores,number of falls and fractures. Mixed-effects models will be performed to compare longitudinal outcome results between groups using intention-to-treat analysis.Ethics and disseminationThe trial was approved by the Northern Sydney Local Health District Human Research Ethics Committee (2022/ETH01794,protocol V.8,dated 03 July 2024). Participants will provide written informed consent prior to inclusion. Findings will be disseminated via peer-reviewed journals,scientific conferences and summary reports to funding bodies.Trial registration numberACTRN12623000867695. View Publication

Segmentation is required to quantify cellular structures in microscopic images. This typically requires their fluorescent labeling. Convolutional neural networks (CNNs) can detect these structures also in only transmitted light images. This eliminates the need for transgenic or dye fluorescent labeling,frees up imaging channels,reduces phototoxicity and speeds up imaging. However,this approach currently requires optimized experimental conditions and computational specialists. Here,we introduce “aiSEGcell” a user-friendly CNN-based software to segment nuclei and cells in bright field images. We extensively evaluated it for nucleus segmentation in different primary cell types in 2D cultures from different imaging modalities in hand-curated published and novel imaging data sets. We provide this curated ground-truth data with 1.1 million nuclei in 20,000 images. aiSEGcell accurately segments nuclei from even challenging bright field images,very similar to manual segmentation. It retains biologically relevant information,e.g. for demanding quantification of noisy biosensors reporting signaling pathway activity dynamics. aiSEGcell is readily adaptable to new use cases with only 32 images required for retraining. aiSEGcell is accessible through both a command line,and a napari graphical user interface. It is agnostic to computational environments and does not require user expert coding experience. Author summaryFluorescence microscopy is the most widely used method to monitor cellular structures in space and time. Fluorescently labeling cellular structures is typically required to localize (“segment”) them in electronic images for subsequent quantification. Deep learning approaches can detect these structures also in only bright field images. This eliminates the need for a fluorescent label,frees up imaging channels,speeds up imaging,and reduces the harmful effects of exposing cells to high intensity light. However,label free segmentation currently requires optimized experimental conditions and computational specialists. Therefore,we developed “aiSEGcell” a user-friendly deep learning-based software to segment nuclei and cells in only bright field images. We extensively evaluated aiSEGcell on different common experimental conditions and showed that biologically even sensitive relevant information is retained. Furthermore,we demonstrated that aiSEGcell is adaptable by retraining to new applications with very little required data. We make it accessible for users with no required expert coding experience in a wide range of computational environments. Finally,we openly share our very large imaging data sets to further the development of other segmentation approaches. View Publication

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS),but GWAS do not reveal causal variants,effector genes,or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C,Hi-C,ATAC-seq,and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features,and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific,but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types,suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However,trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this,we pharmacologically validated squalene synthase,a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE,as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms,and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression,function,and pathology across multiple,distinct tissues and cell types. View Publication

T cell-derived cancers are hallmarked by heterogeneity,aggressiveness,and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here,we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept,we identified an antibody with unique specificity toward a distinct T cell receptor (TCR) and developed antibody-drug conjugates,precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization,associated with induction of cancer cell death with strong signs of apoptosis. Furthermore,cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules’ anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge,this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies. Graphical abstract Harald Kolmar and colleagues report a novel approach for the treatment of the difficult-to-treat T cell lymphoma/leukemia based on targeting the clonally rearranged T cell receptor expressed by the malignant T cell population. The developed antibody-drug conjugates precisely eliminate target T cells while preserving the integrity of the T cell repertoire and cellular immunity. View Publication

Clinical studies have shown that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is associated with aggressive periodontitis and can potentially trigger or exacerbate rheumatoid arthritis (RA). However,the mechanism is poorly understood. Here,we show that systemic infection with A. actinomycetemcomitans triggers the progression of arthritis in mice anti-collagen antibody-induced arthritis (CAIA) model following IL-1β secretion and cell infiltration in paws in a manner that is dependent on caspase-11-mediated inflammasome activation in macrophages. The administration of polymyxin B (PMB),chloroquine,and anti-CD11b antibody suppressed inflammasome activation in macrophages and arthritis in mice,suggesting that the recognition of lipopolysaccharide (LPS) in the cytosol after bacterial degradation by lysosomes and invasion via CD11b are needed to trigger arthritis following inflammasome activation in macrophages. These data reveal that the inhibition of caspase-11-mediated inflammasome activation potentiates aggravation of RA induced by infection with A. actinomycetemcomitans. This work highlights how RA can be progressed by inflammasome activation as a result of periodontitis-associated bacterial infection and discusses the mechanism of inflammasome activation in response to infection with A. actinomycetemcomitans. View Publication

IntroductionThe early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins,including the E3/49K protein,which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45,thereby modulating activation of T and NK cells.MethodsConsidering that E3/49K represents the most polymorphic viral protein among species D HAdVs,we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus,this feature is conserved regardless of the pathological associations of the respective HAdV types.ResultsIt appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover,E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45,suggesting E3/49K as a potential tool to investigate CD45 specific functions.ConclusionWe identified B cells as new direct target of E3/49K-mediated immune modulation,representing a novel viral immunosubversive mechanism. View Publication

AbstractChromobox 2 (CBX2),an epigenetic reader and component of polycomb repressor complex 1,is highly expressed in >75% of high-grade serous carcinoma. Increased CBX2 expression is associated with poorer survival,whereas CBX2 knockdown leads to improved chemotherapy sensitivity. In a high-grade serous carcinoma immune-competent murine model,knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME),understanding that the TIME plays a critical role in disease progression and development of therapy resistance. Exploration of existing datasets demonstrated that elevated CBX2 expression significantly correlated with specific immune cell types in the TIME. RNA sequencing and pathway analysis of differentially expressed genes demonstrated immune signature enrichment. Confocal microscopy and co-culture experiments found that modulation of CBX2 leads to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2-overexpressing cells showed increased M2-like macrophages and decreased phagocytosis activity. Cbx2 knockdown in the Trp53-null,Brca2-null ID8 syngeneic murine model (ID8 Trp53−/−Brca2−/−) led to decreased tumor progression compared with the control. NanoString immuno-oncology panel analysis suggested that knockdown in Cbx2 shifts immune cell composition,with an increase in macrophages. Multispectral immunohistochemistry (mIHC) further confirmed an increase in macrophage infiltration. Increased CBX2 expression leads to recruitment and polarization of protumor macrophages,and targeting CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies.Significance:CBX2 expression correlates with the TIME. CBX2 modulation shifts the macrophage population,potentially leading to an immunosuppressive microenvironment,highlighting CBX2 as a target to improve efficacy of immunotherapy. View Publication

mRNA applications have undergone unprecedented applications—from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation,involving high costs,risks,and toxicity,emphasizing the need for alternative way as mRNA technology. We successfully developed,screened,and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells,KHYG-1 cells,and primary NK cells with high efficiency without cytotoxicity,while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover,the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably,the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether,our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies. Graphical abstract Pichon and colleagues developed imidazole lipids-based mRNA platforms very efficient to transfect both NK-92 cells,KHYG-1 cells and primary NK cells without cytotoxicity. They succeeded to replace IL-2 protein by IL-2 mRNA transfection and obtained NK cells with extended viability with preserved biomarkers and full functionalities to kill target cells. View Publication

IntroductionHeme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation,and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors,and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation,that is,NOD-like receptor protein 3 (NLRP3),glutathione (GSH),GSH disulfide (GSSG),and interleukin 6 (IL-6).MethodsThe study population comprised three age groups: young adults (age range,19–23 years),older adults aged under 85 years (age range,73–84 years),and older adults aged over 85 years (age range,85–92 years). NLRP3,GSH,and GSSG concentrations were measured in serum,whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2,lipopolysaccharide (LPS),or phorbol 12-myristate 13-acetate (PMA) with ionomycin.ResultsThe analysis of serum NLRP3,GSH,and GSSG concentrations revealed no statistically significant differences among the studied age groups. However,some typical trends of aging were observed,such as a decrease in GSH concentration and an increase in both GSSG level,and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover,statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells.ConclusionsThese results showed that NK cells can express HO-1 at a basal level,which was significantly increased in activated cells,even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters. View Publication

Background & AimsType 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However,little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model,Meta1 gastroids,to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells.MethodsWe performed single-cell RNA sequencing to characterize Meta1 gastroids,which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation.ResultsMeta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids.ConclusionsIL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration. Graphical Abstract View Publication