技术资料

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

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

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

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

Macrophages play vital roles in innate and adaptive immunity,and their functions are mediated via phagocytosis and antigen presentation. Despite the effort to identify phagocytic checkpoints and explore their mechanism of action,current checkpoint-scanning strategies cannot provide a complete and systematic list of such immune checkpoints. Here,we perform in vitro phagocytosis assays using primary healthy donor macrophages co-cultured with breast cancer cells followed by ribosome profiling of sorted macrophages,to identify immune system-specific checkpoints. We observe a downregulation of CD37 in phagocytic macrophages and demonstrate that targeting CD37 with a specific antibody promotes the phagocytosis of multiple cancer cells in vitro. Mechanistically,tumorous macrophage migration inhibitory factor (MIF) directly binds to CD37,promoting the phosphorylation of CD37Y13 and activating a transduction cascade that involves the recruitment of SHP1 and inhibition of AKT signaling,ultimately impairing phagocytosis. In vivo,targeting CD37 promotes tumor clearance in multiple preclinical mouse models and synergizes with anti-CD47 therapy. Thus,our study identifies a previously unidentified phagocytic checkpoint and provides new potential for precise therapy. Cancer cells evade the immune system by disrupting phagocytic clearance. Here,the authors identify CD37 as a potential checkpoint molecule expressed on non-phagocytes and propose that binding to tumor-derived MIF reduces the phagocytic ability via inhibiting the AKT pathway. In preclinical mouse models,anti-CD37-based therapy enhances phagocytosis by macrophages,facilitating tumor clearance. View Publication

SummaryAlthough chimeric antigen receptor (CAR) T cells have demonstrated therapeutic activity in hematopoietic malignancies,tumor heterogeneity has impeded the efficacy of CAR T cells and their extension into successful solid tumor treatment. To address these challenges,induced pluripotent stem cell (iPSC)-derived T (iT) cells are engineered to uniformly express CAR and T cell receptor (TCR),enabling targeting of both surface and intracellular antigens,respectively,along with a high-affinity,non-cleavable variant of CD16a (hnCD16) to support antibody-dependent cellular cytotoxicity (ADCC) when combined with therapeutic antibodies. Co-expression of each antitumor strategy on engineered iT cells enables independent and antigen-specific targeting across a diverse set of liquid and solid tumors. In heterogeneous tumor models,coactivation of these modalities is required for measurable antitumor efficacy,with activation of all three modalities displaying maximal efficacy. These data highlight the therapeutic potential of an off-the-shelf engineered iPSC-derived trimodal T cell expressing CAR,TCR,and hnCD16 to combat difficult-to-treat heterogeneous tumors. Graphical abstract Highlights•CAR,TCR,and hnCD16 can be uniformly co-expressed and can function in iT cells•hnCD16 signals through CD3ζ and arms iT cells with targeting flexibility through ADCC•Concurring CAR,TCR,and hnCD16 activation demonstrates a cooperative effect•Multi-targeting with trimodal iT cells can control heterogeneous tumors in vivo Yang et al. show that (1) trimodal iPSC cells expressing CAR,TCR,and hnCD16 can commit to T cell lineage,(2) hnCD16 signals through CD3ζ in iT cells and arms iT cells with ADCC targeting flexibility,and (3) trimodal iT cells control antigen-heterogeneous tumors in vivo through multi-modal targeting. View Publication

Staphylococcus aureus is a notorious human pathogen that thrives in macrophages. It resides in mature phagolysosomes,where a subset of the bacteria eventually begin to proliferate. How S. aureus acquires essential nutrients,such as amino acids,for growth in this niche is poorly understood. Using a long-term primary human macrophage infection model,we show that branched-chain amino acid (BCAA) uptake mediated by the major transporter BrnQ1 is required by S. aureus for intracellular replication in macrophages and we provide mechanistic insight into the role of BCAAs in the success of intracellular S. aureus. Loss of BrnQ1 function renders intracellular S. aureus non-replicative and non-cytotoxic. The defective intracellular growth of S. aureus brnQ1 mutants can be rescued by supplementation with BCAAs or by overexpression of the BCAA transporters BrnQ1 or BcaP. Inactivation of the CodY repressor rescues the ability of S. aureus brnQ1 mutants to proliferate intracellularly independent of endogenous BCAA synthesis but dependent on BcaP expression. Non-replicating brnQ1 mutants in primary human macrophages become metabolically quiescent and display aberrant gene expression marked by failure to respond to intraphagosomal iron starvation. The bacteria remain,however,viable for an inordinate length of time. This dormant,yet viable bacterial state is distinct from classical persisters and small colony variants. Author summaryStaphylococcus aureus is a prominent human pathogen causing acute and chronic disease. It is facultatively intracellular and can reside within many host cell types,including professional phagocytes such as macrophages. The intracellular state contributes to dissemination,recurrence and infection chronicity. Chronic and relapsing infections are often associated with so-called persister phenotypes. Growth arrest and metabolic quiescence,accompanied by antibiotic tolerance,are hallmarks of persistence in bacteria. Antibiotic pressure is a major factor in triggering intracellular persistence. The small colony variant (SCV),an extensively studied form of S. aureus persister,can arise in the absence of antibiotic pressure and exhibits very distinctive phenotypic characteristics.Here,we describe a different growth-arrested state of S. aureus,which conforms to the definition of a non-antibiotic-driven form of intracellular dormancy,triggered by branched-chain amino acid starvation in macrophages. We show that loss of function of the major branched-chain amino acid transporter BrnQ1 renders intracellular S. aureus non-replicative and metabolically quiescent for an inordinate period of time. Upon stochastic exit from infected macrophages,brnQ1 mutants retain full virulence. This dormancy differs from classical persistence or SCVs and uncovers an underestimated role for BCAA uptake in the success of intracellular S. aureus. View Publication

AbstractBackgroundAcute graft‐versus‐host disease (aGVHD) arises from the imbalance of host T cells. Galectin‐9 negatively regulates CD4 effector T cell (Th1 and Th17) function by binding to Tim‐3. However,the relationship between Galectin‐9/Tim‐3 and CD4+ T subsets in patients with aGVHD after Haplo‐HSCT (haploidentical peripheral blood hematopoietic stem cell transplantation) has not been fully elucidated. Here,we investigated the role of Galectin‐9 and CD4+T subsets in aGVHD after haplo‐HSCT.MethodsForty‐two patients underwent Haplo‐HSCT (26 without aGVHD and 16 with aGVHD),and 20 healthy controls were included. The concentrations of Galectin‐9,interferon‐gamma (IFN‐γ),interleukin (IL)‐4,transforming growth factor (TGF)‐β,and IL‐17 in the serum and culture supernatant were measured using enzyme‐linked immunosorbent assay or cytometric bead array. The expression levels of Galectin‐9,PI3K,p‐PI3K,and p‐mTOR protein were detected by western blot analysis. Flow cytometry was used to analyze the proportions of CD4+ T cell subsets. Bioinformatics analysis was performed.ResultsIn patients with aGVHD,regulatory T (Treg) cells and Galectin‐9 decreased,and the Th1,Th17,and Treg cells were significantly imbalanced. Moreover,Treg and Galectin‐9 were rapidly reconstituted in the early stage of patients without aGVHD after Haplo‐HSCT,but Th17 cells were reconstituted slowly. Furthermore,Tim‐3 upregulation on Th17 and Th1 cells was associated with excessive activation of the PI3K/AKT pathway in patients with aGVHD. Specifically,in vitro treatment with Galectin‐9 reduced IFN‐γ and IL‐17 production while augmenting TGF‐β secretion. Bioinformatics analysis suggested the potential involvement of the PI3K/AKT/mTOR pathway in aGVHD. Mechanistically,exogenous Galectin‐9 was found to mitigate aGVHD by restoring the Treg/Teffs (effector T cells) balance and suppressing PI3K.ConclusionGalectin‐9 may ameliorate aGVHD after haplo‐HSCT by modulating Treg/Teffs balance and regulating the PI3K/AKT/mTOR pathway. Targeting Galectin‐9 may hold potential value for the treatment of aGVHD. In patients with acute graft‐versus‐host disease (aGVHD),the expression of Tim‐3 is significantly increased. Galectin‐9 binding to Tim‐3 may inhibit the activation of the PI3K/AKT pathway and enhance the function of Treg cells. On the other hand,transforming growth factor (TGF)‐β promotes the differentiation of Treg cells through autocrine secretion,while TGF‐β induces the expression of Galectin‐9 in a paracrine manner. The increased Treg cells can inhibit the activation of Th1 and Th17 cells by secreting TGF‐β,thus alleviating aGVHD and inducing immune tolerance View Publication

AbstractT cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection,including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT,but current sampling methods require a healthcare provider and are expensive,limiting the ability to study these populations longitudinally. To address these challenges,we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive,self-applied,and low in cost. To demonstrate reproducibility,we sampled the cervicovaginal fluid of healthy,reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells,and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live,CD45+ leukocytes,as well as distinct populations of T cells and B cells. Within the T cell compartment,activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches,including identification of both tissue-resident and migratory populations. In addition,the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies. View Publication

SUMMARYThe mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks supplementation of antiretroviral therapy (ART) with metformin,an indirect mTOR inhibitor used in type-2 diabetes treatment,reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T-cells,together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein,we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T-cells from ART-treated PWH,and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1,metformin decreased virion release,but increased the frequency of productively infected CD4lowHIV-p24+ T-cells. These observations coincided with increased BST2/Tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression,and improved recognition of productively infected T-cells by HIV-1 Envelope antibodies. Thus,metformin exerts pleiotropic effects on post-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH. Graphical Abstract View Publication