技术资料

Cryptococcus gattii is an emerging fungal pathogen that is acquired through the respiratory tract and causes invasive infections in both immunocompromised and otherwise healthy people. Many of these apparently immunocompetent patients are subsequently found to have autoantibodies against the pleiotropic cytokine GM-CSF. In this study,we investigated the potential role of GM-CSF (or CSF2) in the host response to C. gattii using a murine model of infection. Interestingly,infected Csf2-/- mice were found to have significantly improved survival and decreased lung fungal burden compared to wild type (WT) mice. We determined that during C. gattii infection,GM-CSF promotes the differentiation of monocytes into alveolar and interstitial macrophages. When these macrophages are ablated in CCR2-DTR+ mice,there is a corresponding improvement in survival with decreased lung fungal burden,thus phenocopying Csf2-/- mice. WT bone-marrow derived macrophages challenged with C. gattii and interstitial and alveolar macrophages from infected WT mice are unable to undergo M1 polarization,suggesting that monocyte-derived macrophages (moMacs) are rendered permissive for fungal proliferation. Therefore,GM-CSF and moMacs mediate immune responses that are harmful to the host. We further found that GM-CSF and moMacs preferentially promote the influx of eosinophils over neutrophils into the infected lung which is associated with substantial inflammatory lung pathology. Ablation of neutrophils using Mrp8cretg iDTR+ mice significantly increased C. gattii burden in the lungs,indicating that GM-CSF and moMacs block the entry of these beneficial,fungal-clearing granulocytes during infection. Altogether,our results show that GM-CSF plays a key role in impeding host anti-fungal responses to C. gattii by coordinating monocyte-derived macrophages and granulocyte activity and crosstalk. Author summaryCryptococcus gattii is an environmental fungus that can cause severe lung and brain infections after inhalation through the respiratory tract. C. gattii causes disease in patients with known immune deficits but also in individuals that are apparently healthy. Studies on otherwise healthy people who become infected with C. gattii suggest that they may have a previously unrecognized problem involving granulocyte macrophage-colony stimulating factor (GM-CSF),a cytokine,or messenger protein,that is an important part of the immune system. Here,we investigate the role of GM-CSF in the immune response to C. gattii using a mouse model of infection. We find that C. gattii increases GM-CSF in the lungs,leading to the influx of immune cells,including monocyte-derived macrophages and eosinophils,while inhibiting the entry of neutrophils. The macrophages and eosinophils allow the fungus to proliferate and cause inflammatory damage to the lungs,which is ultimately fatal. The absence of neutrophils also contributes to fungal growth,as these immune cells would otherwise be able to help kill the fungus. Our study provides new insight into how GM-CSF regulates immunity to C. gattii and has important implications as to the mechanisms that govern susceptibility to this infection. View Publication

Multi-kilobase knock-ins (KIs) are a necessary,yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI,but the insert size is limited by the viral packaging capacity. Here,we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs,we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets),where relevant genomic regions are enriched via hybridization,achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles,imprecision in the insert and donor concatenation,genotypes of non-KI alleles,and more importantly,uniquely identifies and localizes random integration of the full or partial donor(s). Additionally,the multi-rAAV donor approach is successfully applied to cell lines,including lines intolerant of plasmid DNA,whereas LOCK-seq reliably and efficiently screens for KI clones. Together,the two approaches significantly improve the creation and precision of knock-in models. View Publication

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However,methodologies for isolating functional synaptic structures from these models remain limited. Here,we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air‐liquid interface cerebral organoids (ALI‐COs) at distinct developmental stages (Day 90 and 150). Notably,the method avoids density gradients,requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g.,GAP43) and classical synaptosomal proteins (e.g.,PCLO,BSN,SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane‐enclosed GCPs with fibrous content and mitochondria in Day 90 isolates,and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl‐induced depolarization,which triggered phosphorylation changes in growth cone proteins (GAP43,MARCKS,MARCKSL1),cytoskeletal regulators (DCLK1,SHTN1,MARK4,MAP1B) and protein kinases (CAMK2G,PRKCE) in Day 90 GCPs,as well as classical synaptic vesicle cycle proteins (SYN1,DNM1,RPH3A) at Day 150. Overall,this study establishes a centrifugation‐based protocol for isolating growth cones and immature synapses from human organoids,capturing key stages of synaptic development and enabling scalable,patient‐compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders. Synapses are implicated in several neurological disorders and psychiatric diseases. The emergence and wide use of neural organoids provide a new opportunity to study human synapses in healthy and disease settings. Therefore,we developed a simple method for the enrichment of synaptosomes and growth cone particles from forebrain organoids. The method is based on differential centrifugation,works with small tissue amounts,and is highly reproducible. We validated the functionality of the isolated structures using KCl stimulation and phosphoproteomics. The method enables detailed mapping of protein composition and function during growth cone pathfinding,synaptogenesis,and establishment of neural circuits in organoids. View Publication

Huntington’s disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression,providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However,achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here,we developed allele-selective,CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD,spinocerebellar ataxias types 1 and 3,and dentatorubral pallidoluysian atrophy. The most promising candidate,amiR136-13A,reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover,amiR136-13A reduced the level of HTT1a,a short pathogenic isoform of huntingtin. Collectively,these findings identify amiR136-13A as a potent,selective,and safe therapeutic candidate for HD and potentially other polyQ disorders. View Publication

BACKGROUND: In acute myeloid leukemia (AML),the most sensitive measurable residual disease (MRD) methods are single-gene approaches,but these are applicable only in ~60% of AML cases. METHODS: We applied multi-omics single-cell analysis on diagnostic and first remission samples to identify leukemia-specific molecular markers for subsequent MRD monitoring in six AML patients lacking AML-defining variants. RESULTS: Five selection criteria were defined to identify suitable MRD markers. Markers of primordial leukemic clones were identified by combining data from single-cell sequencing and immunophenotyping. Specific markers suitable for use in MRD follow-up were identified in 6/6 patients,in some cases in myelodysplasia-related genes and clonal hematopoiesis-related genes usually not recommended for use in MRD determinations. Patient-specific ddPCR (limits of detection: 0.06-0.0011%) or EC-NGS assays correlated with therapeutic responses: 0/4 markers displayed molecular relapses in three non-relapsing patients,contrary to 4/4 markers of three relapsing patients. Of these,3/4 and 1/4 markers detected molecular relapses earlier than or simultaneous with conventional methods,respectively (-115 to -338 days). CONCLUSIONS: Our results demonstrate that single-cell subclonal mapping at diagnosis and during first remission enables selection of reliable MRD targets for personalized disease surveillance in patients lacking conventional MRD markers. View Publication

Byrd,Marmorale and colleagues conducted a yeast genome-wide screen to identify genes affecting TDP-43 clearance in ALS cell models. They found that ESCRT complex genes and the ubiquitin ligase Rsp5/NEDD4 facilitate TDP-43 endolysosomal clearance. TDP-43 accumulation also induces giant MVB-like organelles. A pathological hallmark in >97% of amyotrophic lateral sclerosis (ALS) cases is the cytoplasmic mislocalization and aggregation of TDP-43,a nuclear RNA-binding protein,in motor neurons. Driving clearance of cytoplasmic TDP-43 reduces toxicity in ALS models,though how TDP-43 clearance is regulated remains controversial. We conducted an unbiased yeast screen using high-throughput dot blotting to identify genes that affect TDP-43 levels. We identified ESCRT complex genes,which induce membrane invagination (particularly at multivesicular bodies; MVBs) and genes linked to K63 ubiquitination (particularly cofactors of the E3 ubiquitin ligase Rsp5; NEDD4 in humans),as drivers of TDP-43 endolysosomal clearance. TDP-43 colocalized and bound Rsp5/NEDD4 and ESCRT proteins,and perturbations to either increased TDP-43 aggregation,stability,and toxicity. NEDD4 also ubiquitinates TDP-43. Lastly,TDP-43 accumulation induces giant MVB-like vesicles,within which TDP-43 accumulates in a NEDD4-dependent manner. Our studies shed light on endolysosomal-mediated cytoplasmic protein clearance,a poorly understood proteostasis mechanism,which may help identify novel ALS therapeutic strategies. View Publication

ObjectiveThis study investigated how cumulative environmental exposures influence offspring behaviour and inflammation-related molecular signatures in the brain and peripheral immune system.MethodsA novel "triple-hit" mouse model was developed using C57Bl/6JAusB mice (N = 70),combining preconceptual social stress,antenatal high-fat diet,and a postnatal immune challenge (poly(I:C),10 mg/kg). At 12 weeks,offspring underwent behavioural tests relevant to neurodevelopmental disorders (NDDs),including the Elevated Plus Maze,3-Chamber Social Preference,Self-Grooming,and Marble Burying. A composite NDD-risk index was calculated. Single-cell RNA sequencing (scRNA-seq) and bulk proteomics were performed on male triple-hit offspring to identify differentially expressed genes and proteins associated with inflammatory pathways.ResultsMale triple-hit offspring showed elevated NDD-related behavioural risk and social deficits,not observed in females. scRNA-seq revealed altered inflammatory and ribosomal pathways in brain glia and peripheral immune cells. Proteomic analysis showed decreased abundance of proteins involved in inflammation,translation,chromatin remodelling,and synaptic function in both brain and blood.ConclusionCombined environmental stressors may drive male-specific behavioural and inflammatory changes relevant to NDDs. The identification of overlapping inflammatory signatures in brain and peripheral immune cells supports a role for shared immune mechanisms in brain–immune axis dysfunction. However,these pathway-level findings should be interpreted as preliminary hypotheses and warrant independent validation to confirm their mechanistic significance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-025-02152-y. View Publication

Non-tuberculous mycobacteria (NTM) infections are difficult to cure completely with current treatments,and no specific drugs are available. However,recent reports have indicated that immune checkpoint inhibitors may effectively treat pulmonary NTM infections. In this study,we investigated the expression of immune checkpoint molecules in macrophages,the host cells of NTM,and assessed their impact on the microenvironment of infected lesions. Bulk-RNA sequencing and western blot analyses revealed that macrophages infected with Mycobacterium avium,an NTM species,exhibited a pro-inflammatory phenotype and increased PD-L1 expression. Additionally,immunostaining of an NTM-infected mouse model and human tissues showed that increased PD-L1 expression in macrophages was associated with decreased T cell infiltration and increased T cell exhaustion (upregulated PD-1 expression) within infected lesions. These findings suggest that NTM infections evade cellular immunity by enhancing PD-L1 expression in macrophages. Therefore,PD-L1 inhibition may be a promising therapeutic strategy against NTM infections. View Publication

The clinical efficacy of immune checkpoint inhibitors (ICIs) in colorectal cancer (CRC) remains limited. Modulation of the glucagon-like peptide-1 receptor (GLP-1R) may enhance T-cell-mediated antitumor responses. The present study aimed to evaluate the antitumor effects of the GLP-1R antagonist Exendin 9–39 (Exe-9) combined with anti-programmed cell death protein-1 (PD-1) treatment in preclinical CRC models. Using in vitro co-culture assays,ELISA and in vivo murine models,alongside immunohistochemical and molecular analyses of clinical samples,HT-29 and MC38-OVA colon cancer cell lines were co-cultured in vitro with activated T cells in the presence of Exe-9. In vivo,male BALB/c mice were injected with MC38 to establish a CRC model and nude mice were used to assess T-cell dependency. To evaluate this synergistic effect,BALB/c mice with CRC were treated with Exe-9,anti-PD-1 or a combination. Additionally,clinical CRC samples were analyzed to assess the association of GLP-1R expression with the immunotherapy response. Exe-9 significantly enhanced T-cell-mediated cytotoxicity in CRC cell lines and reduced tumor growth in immunocompetent CRC mice; however,this effect was not observed in nude mice. Furthermore,combination therapy with the GLP-1R antagonist and anti-PD-1 yielded an improved antitumor effect compared with either treatment alone,and high GLP-1R ex2pression in clinical samples correlated with poor ICI response. These findings suggest that GLP-1R antagonism potentiates T-cell-mediated antitumor immunity and may provide a promising adjunctive therapeutic strategy for patients with CRC when combined with ICIs in the future. View Publication

AbstractNatural killer (NK) cell function within tissues extends beyond exerting cytotoxicity,encompassing a range of functions that are just starting to become fully elucidated. In the context of human placentation,NK cells play a key role in enabling initial placentation,which is associated with the acquisition of tolerance-like properties. If and to which extent NK cells maintain these tolerance-like properties over the course of human pregnancy is still poorly understood. We asked if NK cells isolated from the decidual-placental interface of full-term human pregnancies are able to exert effector function. We observed a significant and striking lack in the ability of NK cells isolated from the decidual-placental interface (DPI) to produce interferon-g (IFN-γ) in response to the activating cytokines interleukin (IL)-12,IL-15,and IL-18. In contrast,NK cells from the decidua retained their responsiveness to cytokine-mediated activation. Notably,CD103+CD69+ tissue-resident NK cells were present in both DPI and decidua,yet exhibited distinct effector function from one another. Using high-parameter flow cytometry and single-cell sequencing,we found that this functional discrepancy was not directly predictable based on their cell surface phenotype or cell transcript. Together,our findings reveal the presence of distinct functional resident NK cell populations in 2 anatomically adjacent tissues at healthy full-term pregnancies. View Publication

In vivo,HIV-1 replicates within tissues,yet the impact of three-dimensional (3D) environments on viral spread remains unclear. Our laboratory previously showed that collagen-rich 3D extracellular matrix (ECM) imposes an Environmental Restriction to cell-free Virus Infectivity (ERVI). Here,we demonstrate that ERVI is mediated by adhesive ECM components assembled into tissue-like scaffolds. Transient interactions with collagen fibers rapidly diminish virion infectivity across diverse primary strains by impairing virus fusogenicity. Notably,collagen-experienced particles also induce a distinct antiviral transcriptional program and strong pro-inflammatory cytokine secretion in monocyte-derived macrophages. Mechanistically,collagen contact induces conformational changes in the viral glycoprotein Env,enhances its interaction with toll-like receptor 2 (TLR2),and promotes trafficking into TLR8-positive endosomes,thereby amplifying innate immune sensing. Thus,ERVI functions through a dual mechanism: reducing virion fusogenicity while increasing innate immune detection. These findings identify the biophysical properties of the ECM as a tissue-intrinsic arm of antiviral innate immunity. Collagen based 3D tissue-like cultures have been shown to modulate HIV-1 infectivity and mode of spread. Here,the authors demonstrate that varying the density of type I collagen matrices impair HIV-1 particle fusion and sensitize virions for TLR-based innate immune recognition by macrophages implicating structural changes in Env,TLR2 interaction and TLR8 positive endosomes promoting innate immune detection of viral gRNA. View Publication

Liu et al. develop PRECISE-seq,a single-cell platform linking TCR specificity and avidity to T cell phenotypes in vivo. They reveal that high-potency antiviral T cells become exhausted,while tumor-reactive CD8+ T cells acquire a regulatory Ly49+ state that is restrained by PD-1 blockade,resulting in effector revival within tumors. Linking T cell phenotypes with antigen specificity and functional avidity is critical for understanding in vivo immune responses in infection and cancer. Here,we develop PRECISE-seq,a method that integrates multi-omics T cell analysis with contact-dependent proximity labeling for rapid screening of disease-relevant T cell repertoires,and for linking the relative TCR avidity with T cell phenotypes at single-cell resolution. PRECISE-seq accurately retrieves CMV-specific clonotypes from human peripheral blood and quantitatively measures functional avidity in physiological contexts. We find that high-potency CMV-specific T cells preferentially acquire an exhausted phenotype. In tumors,polyclonal tumor-reactive CD8+ T cells predominantly differentiate into a protumor Ly49+ regulatory state (TLy49),characterized by inhibitory killer cell lectin-like receptor expression and originating from effector memory T cells along a trajectory distinct from exhaustion. Notably,PD-1 blockade reduces TLy49 formation and promotes effector revival,which correlates with responsiveness to immunotherapy. Together,PRECISE-seq enables high-resolution mapping of TCR potency and T cell phenotype,revealing a regulatory axis shaping T cell fate in tumors. Graphical Abstract A multi-part diagram shows workflow linking T-cell antigen specificity,phenotype,PD-1 response mechanisms,and prediction of clinical outcomes.Panel A: An illustration shows the identification of antigen specificity. It includes a depiction of CD 8 cells interacting with antigens and labeled with biotin. Single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) are illustrated with colored bars representing different gene segments. Panel B: A UMAP plot shows the phenotype and TCR potency. The plot is labeled with UMAP_1 and UMAP_2 axes and shows clusters of cells with different TCR potency and exhaustion scores. Panel C: A diagram illustrates the Ly49 plus regulatory state. It shows immune checkpoints,NK receptors,SPP1,granzyme C,and Ly49 genes interacting with a cell. Panel D: A diagram depicting the mechanism of discovery of alpha PD-1 therapy. It shows different types of T cells (T subscript EM,T subscript EFF,T subscript EX,T subscript Ly49) and their responses to alpha PD-1 therapy. Panel E: A diagram predicts clinical response. It shows two figures representing responders and non-responders based on the levels of T subscript EM,T subscript EFF,and T subscript Ly49/KIR cells. View Publication