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ABSTRACTBackgroundIdiopathic pulmonary fibrosis (IPF) is a lethal disease with an unknown etiology and complex pathophysiology that are not fully understood. The disease involves intricate cellular interplay,particularly among various immune cells. Currently,there is no treatment capable of reversing the fibrotic process or aiding lung regeneration. Hepatocyte growth factor (HGF) has demonstrated antifibrotic properties,whereas the adoptive transfer of modified T cells is a well‐established treatment for various malignancies. We aimed to understand the dynamics of T cells in the progression of lung fibrosis and to study the therapeutic benefit of adoptive T cell transfer in a bleomycin‐injured mouse lung (BLM) model.MethodsT cells were isolated from the spleen of naïve mice and transfected in vitro with mouse HGF plasmid and were administered intratracheally to the mice lungs 7 days post‐bleomycin injury to the lung. Lung tissue and bronchoalveolar lavage were collected and analyzed using flow cytometry,histology,qRT‐PCR,ELISA,and hydroxyproline assay.ResultsOur findings demonstrate the successful T cell therapy of bleomycin‐induced lung injury through the adoptive transfer of HGF‐transfected T cells in mice. This treatment resulted in decreased collagen deposition and a balancing of immune cell exhaustion and cytokine homeostasis compared with untreated controls. In vitro testing showed enhanced apoptosis in myofibroblasts induced by HGF‐overexpressing T cells.ConclusionsTaken together,our data highlight the great potential of adoptive T cell transfer as an emerging therapy to counteract lung fibrosis. This study explores the potential of T cells as a therapeutic strategy against idiopathic pulmonary fibrosis (IPF),a progressive lung disease for which there is currently no treatment to reverse fibrosis or restore normal lung function. To investigate an innovative approach using adoptive T cell transfer,T cells isolated from healthy mice were genetically modified to carry a plasmid containing hepatocyte growth factor (HGF). The modified cells were delivered directly into the airways of mice with bleomycin‐induced lung fibrosis. The results showed a significant reduction in fibrotic scarring,improved immune regulation,and increased apoptosis of pathogenic myofibroblasts. These results highlight the potential of HGF‐engineered T cells as a promising therapeutic approach to combat IPF. View Publication

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Macrophages are potential reservoirs of infection,and dissemination to other organ systems is also observed. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle,providing a blueprint for parasite development. While a small population of infected macrophages mount a response,most remain transcriptionally unchanged,suggesting that the majority of parasites may avoid host detection. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together,our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program. Microsporidia such as Encephalitozoon intestinalis are single-celled intracellular parasites that cause opportunistic infections and disease in humans involving infection of macrophages. Here the authors infect human macrophages with E. intestinalis,in vitro and use single cell transcriptomics to assess the consequences of cellular infection compared to bystander effects on macrophages and provide insights into the E. intestinalis developmental program. View Publication

Hermansky-Pudlak Syndrome (HPS) type 1 (HPS-1) is an autosomal recessive disorder characterized by oculocutaneous albinism,platelet dysfunction,and pulmonary fibrosis (HPS-PF),the leading cause of mortality in these patients. HPS-PF manifests earlier than idiopathic pulmonary fibrosis,typically between 30 and 40 years of age. The etiology and drivers of HPS-PF progression remain poorly understood,and no FDA-approved therapies exist. Neutrophil extracellular traps (NETs) and neutrophil-derived mediators have emerged as key players in fibrosis,promoting lung injury,inflammation,and fibroblast activation. This study evaluates the role of neutrophil activation in age-related changes in patients with HPS-1,focusing on differences in inflammatory markers,neutrophil granules,and NETosis capacity. We observed significantly elevated levels of NETs,neutrophil granule proteins (NE,NGAL,LF),and inflammatory cytokines (IL-8,IL-6) in patients with HPS-1 older than 40 years compared to younger patients and healthy controls. Additionally,fibrosis-related markers (MMP-7 and MMP-8) were significantly higher in older patients. Elevated levels of anandamide (AEA),a circulating marker of HPS-PF,were positively associated with neutrophil granule markers in older patients,suggesting its association with fibrosis. Neutrophils from older patients also demonstrated increased NETosis capacity. These findings suggest that age-related neutrophil activation may contribute to an inflammatory environment that promotes fibrosis progression in HPS-1.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13023-025-03758-5. View Publication

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However,CAR T cell products exhibit batch-to-batch variability in cell number,quality,and in vivo efficacy due to donor-to-donor heterogeneity,and pre/post-manufacturing processes,and the manufacturing of such products necessitates careful testing,both post-manufacturing and pre-infusion. Here,we introduce the Cell Trajectory Modulation (CTM) assay,a microfluidic,label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.e.,size,deformability). CTM assay correlates with phenotypic metrics,including CD4:CD8 ratio,memory subtypes,and cytotoxic activity. Validated across multiple donors and culture platforms,the CTM assay requires fewer than 10,000 cells and delivers results within 10 minutes. Compared to labeled flow cytometry processing,the CTM assay offers real-time data to guide adaptive manufacturing workflows. Thus,the CTM assay offers an improvement over existing phenotypic assessments,marking a step forward in advancing CAR T cell therapy manufacturing. CAR T cell manufacturing faces significant challenges that impact cell quality and in vivo efficacy. This necessitates reliable cellular characterization methods. Here the authors present a real-time,label-free,microfluidic method that profiles cellular biophysical properties and correlates them to activation state and CAR T potency,facilitating the rapid phenotypic cell assessment during production. View Publication

Despite the success of chimeric antigen receptor T (CART) cell therapy in hematological malignancies,durable remissions remain low. Here,we report CART senescence as a potential resistance mechanism in 41BB-costimulated CART cell therapy. To mimic cancer relapse,we utilized an in vitro model with repeated CART cell activation cycles followed by rest periods. Using CD19-targeted CART cells with costimulation via 4-1BB-CD3ζ (BBζ) or CD28-CD3ζ (28ζ),we showed that CART cells undergo functional,phenotypical,and transcriptomic changes of senescence,which is more prominent in BBζ. We then utilized two additional independent strategies to induce senescence through MYC activation and irradiation. Induction of senescence impaired BBζ activity but improved 28ζ activity in preclinical studies. These findings were supported by analyses of independent patient data sets; senescence signatures in CART cell products were associated with non-response to BBζ but with improved clinical outcomes in 28ζ treatment. In summary,our study identifies senescence as a potential mechanism of failure predominantly in 41BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. SignificanceWe identified senescence as a cause of failure in CART cell therapy,predominantly in 4-1BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. View Publication

BackgroundLymphatics provide a route for breast cancer cells to metastasize. Lymphatic endothelial cells (LECs),which form the structure of lymphatic vessels,play a key role in this process. Although LECs are pivotal in cancer progression,studies often rely on commercially available cell lines that may not accurately reflect the tumor microenvironment. Therefore,there is a pressing need to directly study patient-derived LECs to better understand their role in breast cancer.MethodsThis study developed a method to isolate and characterize LECs directly from human breast-to-axilla adipose tissue. We used magnetic cell separation to remove CD45 + leukocytes and fluorescence-activated cell sorting to isolate cells expressing CD31 and podoplanin. Isolated cells were cultured under conditions promoting endothelial cell growth and were characterized through various assays assessing proliferation,tube formation,and gene expression patterns.ResultsThe sorted CD31 + /PDPN + /CD45 − cell populations exhibited marked increases in proliferation upon VEGF-C stimulation and formed tubule structures on BME-coated dishes,confirming their LEC properties. Notably,isolated LECs showed distinct gene expression patterns depending on the presence of lymph node metastasis and lymphatic invasion.ConclusionsThe ability to isolate and characterize patient-derived LECs from mammary adipose tissue offers new insights into the cellular mechanisms underlying breast cancer metastasis. Significant gene expression variability related to disease state highlights the potential of these cells as biomarkers and therapeutic targets. This study emphasizes the importance of using patient-derived cells to accurately assess the tumor microenvironment,potentially leading to more personalized therapeutic approaches.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-025-02067-w. View Publication

ABSTRACTCytotoxicity is a cornerstone of immune defense,critical for combating tumors and infections. This process relies on the coordinated action of granzymes and pore‐forming proteins,with granzyme B (GZMB) and perforin (PRF1) being key markers and the most widely studied molecules pertaining to cytotoxicity. However,other human granzymes and cytotoxic components remain underexplored,despite growing evidence of their distinct,context‐dependent roles. Natural killer cell granule protein 7 (NKG7) has recently emerged as a crucial cytotoxicity regulator,yet its expression patterns and function are poorly understood. Using large publicly available single‐cell RNA sequencing atlases,we performed a comprehensive profiling of cytotoxicity across immune subsets and tissues. Our analysis highlights NKG7 expression as a strong marker of cytotoxicity,exhibiting a strong correlation with overall cytotoxic activity (r = 0.97) and surpassing traditional markers such as granzyme B and perforin in reliability. Furthermore,NKG7 expression is notably consistent across diverse immune subsets and tissues,reinforcing its versatility and robustness as a cytotoxicity marker. These findings position NKG7 as an invaluable tool for evaluating immune responses and a reliable indicator of cytotoxic functionality across biological and clinical contexts. Cytotoxicity‐associated genes exhibit heterogeneity at the cellular and tissue levels (left). NKG7 gene expression is strongly associated with a cytotoxic gene signature (middle). NKG7 expression is stable and consistently detected in cells across immunologically relevant tissues and within tumor‐infiltrating immune cells (right). Figure generated in collaboration with Susan Stone (https://www.sue‐stone.com). View Publication

Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people and decreased iron availability impairs immunity. Nevertheless,how iron deprivation impacts immune cell function remains poorly characterised. We interrogate how physiologically low iron availability affects CD8+ T cell metabolism and function,using multi-omic and metabolic labelling approaches. Iron limitation does not substantially alter initial post-activation increases in cell size and CD25 upregulation. However,low iron profoundly stalls proliferation (without influencing cell viability),alters histone methylation status,gene expression,and disrupts mitochondrial membrane potential. Glucose and glutamine metabolism in the TCA cycle is limited and partially reverses to a reductive trajectory. Previous studies identified mitochondria-derived aspartate as crucial for proliferation of transformed cells. Despite aberrant TCA cycling,aspartate is increased in stalled iron deficient CD8+ T cells but is not utilised for nucleotide synthesis,likely due to trapping within depolarised mitochondria. Exogenous aspartate markedly rescues expansion and some functions of severely iron-deficient CD8+ T cells. Overall,iron scarcity creates a mitochondrial-located metabolic bottleneck,which is bypassed by supplying inhibited biochemical processes with aspartate. These findings reveal molecular consequences of iron deficiency for CD8+ T cell function,providing mechanistic insight into the basis for immune impairment during iron deficiency. Iron has been shown to be necessary for the activation and differentiation of CD8+ T cells. Here the authors investigate changes in CD8+ T cell metabolism in iron limiting conditions and find that aspartate is increased yet downstream nucleotide synthesis is suppressed and addition of exogenous aspartate partially rescues T cell function. View Publication

CD8+ T cell exhaustion (Tex) limits immune control of cancer,but the underlying molecular drivers are unclear. In the present study,we identified the prostaglandin I2 (prostacyclin) receptor PTGIR as a cell-intrinsic regulator of T cell exhaustion. Transcriptomic profiling of terminally exhausted (Ttex) CD8+ T cells revealed increased activation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response pathway. Enhancing NRF2 activity (by conditional deletion of Kelch-like ECH-associated protein 1 (KEAP1)) boosts glutathione production in CD8+ T cells but accelerates terminal exhaustion. NRF2 upregulates PTGIR expression in CD8+ T cells. Silencing PTGIR expression enhances T cell effector function (that is,interferon-γ and granzyme production) and limits Ttex cell development in chronic infection and cancer models. Mechanistically,PTGIR signaling impairs T cell metabolism and cytokine production while inducing transcriptional features of Tex cells. These findings identify PTGIR as a NRF2-dependent immune checkpoint that regulates balance between effector and exhausted CD8+ T cell states. Targeting CD8+ T cell exhaustion is a strategy to enhance immune checkpoint inhibition and to fight cancer. Here the authors show a NRF2-dependent role for the prostaglandin I2 receptor PTGIR in controlling T cell exhaustion. View Publication

Achieving a cure is an urgent need for patients with advanced solid tumors. Here,we discover that oncolytic virus (OV) infection enhances IL-18 receptor expression but fails to increase IL-18 ligand expression. Therefore,we engineer armed oncolytic alphavirus M1 expressing wild-type IL-18 (wtIL-18) or a mutant variant (mutIL-18) that evades IL-18 binding protein (IL-18BP) while maintaining IL-18 receptor (IL-18R) binding. Intravenous administration of M1-mutIL-18 suppresses the growth of multiple advanced solid tumors in C57BL/6 and BALB/c mouse models and promotes long-term systemic immune memory. Mechanistically,armed M1-mutIL-18 enhances directed clonal expansion and differentiation of CD8+ T cells and sustains IFN-γ production. Thus,armed M1-mutIL-18 promotes dendritic cell (DC) activation,priming and activation of CD8+ T cells in lymphatic organs,and infiltration of IL-18R+ CD8+ T cells in the tumor microenvironment,establishing a positive feedback loop. We further show that a PD-L1 inhibitor enhances the anti-tumor efficacy of mutIL-18 OVs. These results highlight the importance of the IL-18 pathway in oncolytic virus therapy and implicate reprogramming ligand-receptor interaction as an effective strategy for immunotherapy. Immunotherapy holds great potential,although strategies for durable responses against solid tumors are still needed. Here,the authors combine oncolytic virus (OV) engineering and reprogramming of the IL-18 pathway,showing that armed OVs expressing a decoy-resistant IL-18 elicit anti-tumor immunity and long-term immunological memory against multiple refractory tumors in mice. View Publication

Imaging macrophage trafficking in solid tumors has major implications for cancer diagnosis,prognosis,and therapy. Here,we show that macrophage labeling with lipid-shelled microbubbles enables ultrasound imaging at single-cell level. Crucially,microbubble labeling and sonication at low mechanical indexes do not affect macrophage viability,migration,phenotype,and cytokine secretion profile,supporting the notion that ultrasound imaging can be used for nondestructive macrophage imaging. Despite the damping exerted on the microbubble oscillations by the cellular compartments,the microbubbles exhibit highly nonlinear behavior upon sonication,allowing for high specificity nonlinear US imaging under in vitro and in vivo conditions. Subsequently,we demonstrate that nonlinear ultrasound imaging can selectively monitor macrophage accumulation and extravasation in solid tumors in rodents for at least 8 h after intravenous administration. These findings establish ultrasound as a noninvasive platform for immune cell trafficking in solid tumors and highlight its potential to advance cancer diagnosis,monitoring,and therapy. Imaging macrophage trafficking in solid tumors has implications for cancer diagnosis and prognosis. by labeling macrophages with lipid-shelled microbubbles in combination with ultrasound,the authors here achieve nondestructive in vivo intravenously administrated macrophage imaging at single cell level with 100 µm resolution till 8 h in solid tumors in rodents. View Publication

Menopause not only affects fertility but also has widespread impact on systemic health. Yet,the molecular mechanisms underlying this process are not fully understood,partly due to the absence of robust,age-relevant preclinical models with comprehensive molecular and phenotypic characterization. To address this,we systematically compared three candidate mouse models of menopause: (1) intact aging,(2) chemical ovarian follicle depletion using 4-vinylcyclohexene diepoxide (VCD) administered at multiple ages,and (3) Foxl2 haploinsufficiency,a genetic model based on a transcription factor linked to human premature ovarian failure. Through histology,serum hormone profiling,single-cell transcriptomics and machine-learning approaches,we uncovered both shared and model-specific features of follicle loss,endocrine disruption,and transcriptional remodeling. The VCD and Foxl2 haploinsufficiency models revealed distinct patterns of hormonal and immune alterations not captured by intact aging alone. This comparative framework enables informed selection of context-appropriate preclinical rodent models to study menopause and the broader physiological consequences of ovarian aging. View Publication