技术资料

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here,we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1–2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients,with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states,including a naive-like state,but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively,these data support the feasibility,safety,and immunogenicity of this treatment in resected NSCLC. View Publication

Johnson,Ogishi,and Domingo-Vila et al. describe two siblings with inherited PD-L1 deficiency. Human PD-L1 deficiency underlies early-onset T1D,like PD-1 deficiency,but does not lead to fatal autoimmunity with extensive leukocytic dysregulation,unlike PD-1 deficiency. View Publication

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 ( V606M ; R453C ),MYBPC3 ( G148R ) or digenic variants ( MYBPC3 P955fs,TNNI3 A157V ). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls,variant-positive CMs show sarcomere disorganization,higher contractility,calcium transients,and ATPase activity. However,only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM. View Publication

SARS-CoV-2 Omicron variant has evolved into sublineages. Here,we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However,there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population. Subject areas: Immunology,Immune response,Virology View Publication

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however,an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4 + MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity,but MP cells retain less compromised mitochondria compared to effector CD4 + T cells,and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells. Subject areas: immunity,cell biology View Publication

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem‐like cancer cells,called glioblastoma stem cells (GSCs). GSCs have the capacity for self‐renewal and tumorigenesis,leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem‐like cancer cells,their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation,novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image‐based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs,facilitating the development of NK cell‐based immunotherapy for improved GBM treatment. View Publication

Neural–tumor interactions drive glioma growth as evidenced in preclinical models,but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients’ survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma,primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like,astrocyte-like and oligodendrocyte-progenitor-like,alongside oligodendrocytes and excitatory neurons. In line with these findings,high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients,a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients’ plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes. Subject terms: Translational research,CNS cancer,DNA methylation View Publication

Of all gynecologic cancers,epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts,90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa,we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover,treatment with LIFR inhibitor,EC359 significantly reduced OCa cell viability and cell survival with an IC 50 ranging from 5-50 nM. Furthermore,EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro,ex vivo and in vivo models including cell-based xenografts,patient-derived explants,organoids,and xenograft tumors,we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally,EC359 therapy considerably improved tumor immunogenicity by robust CD45 + leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-,B-,and other immune cells. Collectively,our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa. Subject terms: Molecular medicine,Ovarian cancer View Publication

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here,we report a novel antigen Plasmodium prohibitin “ Pf PHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach,we show that loss of Hsp70A1A in red blood cells (RBCs) or Pf PHB2 in infected red blood cells (iRBCs),respectively,inhibit Pf PHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting Pf PHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant Pf PHB2 binds to RBCs which is inhibited by anti- Pf PHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of Pf PHB2 to serve as antigen is further supported by detection of anti- Pf PHB2 antibody in patient sera. Overall,this study proposes Pf PHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment. Subject areas: biochemistry,molecular biology,immunology View Publication

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock,but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma,we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction,abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent,synergistic therapeutic potential agnostic of glioma subtype,leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy,opening avenues for combinatorial treatment in these lethal malignancies. Subject terms: CNS cancer,Paediatric cancer,Radiotherapy View Publication

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs 1 – 3 . Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast,restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness,such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses,whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover,sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion. Subject terms: Haematopoietic stem cells,Self-renewal,Stem-cell niche,Endocytosis,Growth factor signalling View Publication

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here,we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19,the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation. View Publication