技术资料

IntroductionVaccination against COVID-19 has been pivotal in reducing the global burden of the disease. However,Phase III trial results and observational studies underscore differences in efficacy across vaccine technologies and dosing regimens. Notably,mRNA vaccines have exhibited superior effectiveness compared to Adenovirus (AdV) vaccines,especially with extended dosing intervals. MethodsUsing in-host mechanistic modelling,this study elucidates these variations and unravels the biological mechanisms shaping the immune responses at the cellular level. We used data on the change in memory B cells,plasmablasts,and antibody titres after the second dose of a COVID-19 vaccine for Australian healthcare workers. Alongside this dataset,we constructed a kinetic model of humoral immunity which jointly captured the dynamics of multiple immune markers,and integrated hierarchical effects into this kinetics model,including age,dosing schedule,and vaccine type.ResultsOur analysis estimated that mRNA vaccines induced 2.1 times higher memory B cell proliferation than AdV vaccines after adjusting for age,interval between doses and priming dose. Additionally,extending the duration between the second vaccine dose and priming dose beyond 28 days boosted neutralising antibody production per plasmablast concentration by 30%. We also found that antibody responses after the second dose were more persistent when mRNA vaccines were used over AdV vaccines and for longer dosing regimens. DiscussionReconstructing in-host kinetics in response to vaccination could help optimise vaccine dosing regimens,improve vaccine efficacy in different population groups,and inform the design of future vaccines for enhanced protection against emerging pathogens. View Publication

Diffuse intrinsic pontine glioma (DIPG),a lethal pediatric cancer driven by H3K27M oncohistones,exhibits aberrant epigenetic regulation and stem-like cell states. Here,we uncover an axis involving H3.3K27M oncohistones,CREB5/ID1,which sustains the stem-like state of DIPG cells,promoting malignancy. We demonstrate that CREB5 mediates elevated ID1 levels in the H3.3K27M/ACVR1WT subtype,promoting tumor growth; while BMP signaling regulates this process in the H3.1K27M/ACVR1MUT subtype. Furthermore,we reveal that H3.3K27M directly enhances CREB5 expression by reshaping the H3K27me3 landscape at the CREB5 locus,particularly at super-enhancer regions. Additionally,we elucidate the collaboration between CREB5 and BRG1,the SWI/SNF chromatin remodeling complex catalytic subunit,in driving oncogenic transcriptional changes in H3.3K27M DIPG. Intriguingly,disrupting CREB5 super-enhancers with ABBV-075 significantly reduces its expression and inhibits H3.3K27M DIPG tumor growth. Combined treatment with ABBV-075 and a BRG1 inhibitor presents a promising therapeutic strategy for clinical translation in H3.3K27M DIPG treatment. Diffuse intrinsic pontine glioma (DIPG) is driven by H3K27M oncohistones,but the exact related mechanisms are poorly understood. Here,the authors identify an H3.3K27M oncohistone-driven CREB5/ID1 axis that promotes transcriptional changes and progression in DIPG,which could represent a potential therapeutic target. View Publication

Hepatocellular carcinoma (HCC) harbors two types of stem cells—epithelial and mesenchymal stem cells. The mechanism by which epithelial EpCAM-positive HCC cells transform into mesenchymal CD90-positive HCC cells remains unclear. On peritumoral fibrotic nodules,epithelial HCC cells form communities with stromal cells,driving tumor growth and malignancy. We aimed to clarify the mechanism by which epithelial cell adhesion molecule (EpCAM)-positive HCC cells contribute to the phenotype of mesenchymal CD90-positive HCC cells that metastasize to distant sites by elucidating the interaction between EpCAM-positive HCC cells and fibroblasts. EpCAM-positive CD90-negative epithelial HCC cells (Huh1,Huh7,and HCC cells) were converted into metastasis-prone CD90-positive HCC cells by co-culture with fibroblasts (Lx-2 and Tig3-20). We identified the transcription factor JUNB as responsible for this altered phenotype. We found that the overexpression of JUNB in CD90-negative epithelial HCC cells resulted in significant transformation to mesenchymal CD90-positive HCC in vitro and in vivo,showing metastatic potential to the lungs. In addition,the JUNB expression in EpCAM-positive hepatoma cells was increased by paracrine stimulation with fibroblast-derived TGFb1. This study unravels the mechanism by which fibroblasts aggravate the malignancy of liver cancer,and the results suggest that JUNB may be a target for treating liver cancer metastasis. View Publication

BackgroundMesenchymal-stromal-cell-derived extracellular vesicles (MSC-EVs) play a key role in the paracrine effects of MSC and have demonstrated therapeutic potential in various preclinical models. However,clinical translation is hindered by manufacturing practices relying on planar culture systems,fetal bovine serum (FBS)-supplemented media,and non-scalable,low-purity EV isolation methods that fail to meet dose and safety requirements,underscoring the need for innovative approaches. In this study,we developed a scalable platform to manufacture human MSC-EVs at clinically relevant numbers,integrating continuous collection of EV-enriched conditioned media (CM) using a stirred-tank reactor (STR) under xenogeneic-free conditions and a scalable downstream process.MethodsWharton’s jelly-derived MSC (MSC(WJ)) were expanded using microcarriers in a controlled STR using human platelet lysate (hPL)-supplemented medium. Then,a 3-day EV production stage,featuring continuous harvesting of the CM,was established using a novel serum-/xeno(geneic)-free exosome depleted-hPL supplement. For the isolation of MSC-EVs,a scalable process was implemented by pairing tangential flow filtration and anion exchange chromatography. Isolated MSC-EVs were characterised using nanoparticle tracking analysis,protein and zeta potential quantification,western blot analysis of EV protein markers,transmission electron microscopy and uptake studies of fluorescently labelled-EVs.ResultsThe system sustained the efficient expansion of MSC(WJ),reaching a total of (6.03 ± 0.181) x 107 cells after 7 days,which corresponds to a 30.1 ± 0.740-fold expansion. Upon a 3-day continuous CM harvesting,a total of (2.13 ± 0.301) x 1012 EVs were isolated corresponding to a particle yield factor of (1.26 ± 0.186) x 104 EVs/cell/day. MSC-EVs presented high purity levels ((5.53 ± 1.55) x 109 particles/µg),a homogeneous small size distribution (mean diameter of 115 ± 4.88 nm),a surface charge of -23.4 ± 6.23 mV,positive detection of tetraspanins CD9 and CD63 and syntenin-1 and displayed a typical cup-shaped morphology. MSC-EVs were readily incorporated by endothelial cells and two human breast cancer cell lines.ConclusionsOverall,the scalable and Good Manufacturing Practices (GMP)-compliant platform established herein enabled the reproducible manufacturing of MSC-EVs with high purity and generally accepted characteristics concerning size,protein markers,surface charge,morphology,and cellular internalization,validating its potential for future clinical applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04341-2. View Publication

The human placenta,a unique tumor-like organ,is thought to exhibit rare aneuploidy associated with adverse pregnancy outcomes. Discrepancies in reported aneuploidy prevalence in placentas stem from limitations in modeling and detection methods. Here,we use isogenic trophoblast stem cells (TSCs) derived from both naïve and primed human pluripotent stem cells (hPSCs) to reveal the spontaneous occurrence of aneuploidy,suggesting chromosomal instability (CIN) as an inherent feature of the trophoblast lineage. We identify potential pathways contributing to the occurrence and tolerance of CIN,such as autophagy,which may support the survival of aneuploid cells. Despite extensive chromosomal abnormalities,TSCs maintain their proliferative and differentiation capacities. These findings are further validated in placentas,where we observe a high prevalence of heterogeneous aneuploidy across trophoblasts,particularly in invasive extravillous trophoblasts. Our study challenges the traditional view of aneuploidy in the placenta and provides insights into the implications of CIN in placental function. Studies have shown that placental aneuploidy is correlated with adverse pregnancy outcomes,though few causative data are available. Here they show that chromosomal instability is an inherent feature of trophoblasts and normal human placentas,without functional compromise,and provide mechanisms for how this damage is tolerated. View Publication

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of spike are not well studied,the entire viral genome is undergoing evolutionary selection,with several variants containing mutations in the central disordered linker region of the nucleocapsid (N) protein. Here,we identify a mutation (G215C),characteristic of the Delta variant,that introduces a novel cysteine into this linker domain,which results in the formation of a more stable N-N dimer. Using reverse genetics,we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background,where it results in significantly increased viral growth both in vitro and in vivo. Mechanistically,we show that the N:G215C mutant has more encapsidation as measured by increased RNA binding to N,N incorporation into virions,and electron microscopy showing that individual virions are larger,with elongated morphologies. Mutations outside the spike protein in SARS-CoV-2,which are likely to impact viral properties,are currently understudied. This study shows that a nucleocapsid protein mutation in the Delta variant enhances viral growth in vitro and in vivo by increasing viral genome encapsidation. View Publication

IntroductionEnhancer of zeste homolog 2 (EZH2) catalyzes H3K27me3,an epigenetic modification linked to gene silencing,and its overexpression contributes to the progression of hematological malignancies. This study compares the efficacy of a conventional EZH2 inhibitor with a PROTAC-based EZH2 degrader in human lymphoma cell lines. Furthermore,we investigate the anti-tumor effects of combining EZH2 degrader with anti-PD-1,an immune checkpoint inhibitor,focusing on immune cell interactions and underlying mechanisms. MethodsThe cytotoxic effects of the EZH2 degrader and EZH2 inhibitor were evaluated in Burkitt’s,B-cell,cutaneous T-cell,and Hodgkin’s lymphoma cell lines. Additionally,the combination therapy of the EZH2 degrader and anti-PD-1 was assessed both in vitro and in a hu-PBMC-CDX mouse model. ResultsWe evaluated the effects of an EZH2 degrader on seven lymphoma cell lines and observed significant reductions in cell viability compared to EZH2 inhibitor,particularly in Burkitt’s lymphoma cell lines. EZH2 degrader treatment reduced EZH2 and c-Myc expression,induced G2/M cell cycle arrest,and increased apoptosis markers,including cleaved caspase-3 and cleaved PARP. Furthermore,Burkitt’s lymphoma is a PD-L1 negative tumor; however,treatment with the EZH2 degrader resulted in a slight increase in PD-L1 expression. Combining EZH2 degrader with anti-PD-1 significantly enhanced anti-tumor effects compared to monotherapy. In vivo studies using a humanized lymphoma mouse model demonstrated a synergistic anti-tumor effect of EZH2 degrader and anti-PD-1,which was attributed to apoptosis-related pathways. DiscussionThese findings aim to provide insights into the therapeutic potential of targeting EZH2 in combination with immune checkpoint inhibitors for improved treatment of lymphomas. View Publication

AbstractStromal cells are critical regulators of hematopoietic stem/progenitor cells and skeletal homeostasis. Although precise systems for functional analysis are critical to investigate mechanistically bone and bone marrow (BM)-derived stromal cells,the establishment of reproducible,highly enriched ex vivo methods for stromal cell isolation,culture and evaluation have been challenging,leading to inconsistent data on stromal cell function. In this work,we carefully tested ex vivo culture of murine stromal cells from BM and bone and discovered abundant and persistent contamination of monocytes and macrophages. We succeeded in establishing highly enriched ex vivo culture system for stromal cells by eliminating persistent monocytes and macrophages using selection against the immunological markers F4/80,Ly6C,and CD45. Transcriptional and functional assays of enriched stromal cell culture revealed differential characteristics of stromal cells from different origins,a dormant signature for bone-derived cells and a highly proliferative progenitor-like signature for BM-derived cells. Monocyte and macrophage contamination reduced signatures of immature stromal cells such as expression levels of SOX9 and CD140a as well as the cells’ ability to support hematopoietic stem and progenitor cells based on our growth factor-free co-culture system of hematopoietic cells and stromal cells followed by in vivo functional assays. The inhibitory effects of macrophages on stromal cells may be explained by their potent production of inflammatory cytokines such as CXCL2,CCL3,and complement factor (C1q) confirmed by protein immunoassay of culture supernatant,as well as the differential contribution of pre-osteoblasts to the stromal cell population. This study highlights the functional diversity of stromal cells depending on the microenvironment of origin while addressing a critical limitation of murine ex vivo systems. Our robust culture system enables the study of isolated stromal cells function as well as the impact of stromal cells-macrophage crosstalk. View Publication

Polymyositis (PM) is a systemic autoimmune disease characterized by muscular inflammatory infiltrates and degeneration. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) contributes to immune tolerance by inhibiting T cell-mediated autoimmunity. Here,we show that a reduced expression of TIGIT in CD4+ T cells from patients with PM promotes these cells’ differentiation into Th1 and Th17 cells,which could be rescued by TIGIT overexpression. Knockout of TIGIT enhances muscle inflammation in a mouse model of experimental autoimmune myositis. Mechanistically,we find that TIGIT deficiency enhances CD28-mediated PI3K/AKT/mTOR co-stimulatory pathway,which promotes glucose oxidation,citrate production,and increased cytosolic acetyl-CoA levels,ultimately inducing epigenetic reprogramming via histone acetylation. Importantly,pharmacological inhibition of histone acetylation suppresses the differentiation of Th1 and Th17 cells,alleviating muscle inflammation. Thus,our findings reveal a mechanism by which TIGIT directly affects the differentiation of Th1 and Th17 T cells through metabolic‒epigenetic reprogramming,with important implications for treating systemic autoimmune diseases. Aberrant activation of autoreactive T cells is a hallmark of autoimmune diseases. Here,by analysing CD4 T cells from patients with polymyositis and mice with experimental autoimmune myositis,the authors show that TIGIT limits the differentiation of inflammatory Th1 and Th17 cells via a metabolic-epigenetic mechanism,which involves the inhibition of CD28-mediated PI3K/AKT/mTOR costimulatory signaling. View Publication

BackgroundAging is the greatest risk factor for breast cancer,and although epithelial cells are the source of carcinomas,epithelial changes alone do not fully explain cancer susceptibility. Fibroblasts and macrophages are key stromal constituents around the cells of origin for cancer in breast tissue. With age,macrophages surrounding terminal ductal lobular units (TDLUs) become increasingly immunosuppressive. CD105+ fibroblasts intercalate within TDLUs,drive luminal differentiation,and give rise to immunosuppressive cancer-associated fibroblasts in other tissues. We propose that differences in fibroblasts are a crucial component of the stroma that shapes cancer susceptibility.MethodsPrimary peri-epithelial fibroblast cultures were established from prophylactic and reduction mammoplasties from 30 women ranging in age from 16 to 70 years and from BRCA1 mutation carriers. Growth characteristics,transcriptional profiles,differentiation potential,and secreted proteins were profiled for fibroblast subtypes from diverse donors. Co-cultures with fibroblasts,macrophages,and T cells were used to ascertain the functional role played by CD105+ fibroblasts in immune cell modulation.ResultsWe found that peri-epithelial CD105+ fibroblasts are enriched in older women as well as women who carry BRCA1 mutations. These CD105+ fibroblasts exhibit robust adipogenesis and secrete factors related to macrophage polarization. Macrophages cocultured with fibroblasts better maintain or enhance polarization states than media alone. CD105+ fibroblasts increased expression of immunosuppressive macrophage genes. CD105+ fibroblasts supported anti-inflammatory macrophage-mediated suppression of T cell proliferation,whereas CD105− fibroblasts significantly reduced the suppressive effect of anti-inflammatory macrophages on T cell proliferation.ConclusionsEstablishment of a coculture system to dissect the molecular circuits between CD105+ fibroblasts and macrophages that drive immunosuppressive macrophage polarization has broad utility in understanding mammary gland development and events that precede cancer initiation. CD105+ fibroblasts and macrophages may coordinate to suppress immunosurveillance and increase breast cancer susceptibility.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-025-02040-7. View Publication

SummaryVitreous infiltrating immune cells represent an accessible tissue sample that can provide valuable information on the intraocular immune response without compromising the structural and functional integrity of the eye. Here,we present our customized protocol for isolating and characterizing vitreous immune cell infiltrates by flow cytometry and single-cell transcriptomics. The protocol includes surgical modifications for sample collection and other measures to maximize the yield of immune cells from human vitreous samples.For complete details on the use and execution of this protocol,see Alam et al.1 Graphical abstract Highlights•Customized protocol for vitreous immune cell isolation in posterior segment uveitis•Antigen-specific activation and intracellular cytokine staining of vitreous T cells•Paired 5′ single-cell gene expression and V(D)J sequencing of vitreous immune cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Vitreous infiltrating immune cells represent an accessible tissue sample that can provide valuable information on the intraocular immune response without compromising the structural and functional integrity of the eye. Here,we present our customized protocol for isolating and characterizing vitreous immune cell infiltrates by flow cytometry and single-cell transcriptomics. The protocol includes surgical modifications for sample collection and other measures to maximize the yield of immune cells from human vitreous samples. View Publication

Antibodies targeting the highly conserved prehairpin intermediate (PHI) of class I viral membrane-fusion proteins are generally weakly neutralizing and are not considered viable therapeutic agents. We previously demonstrated that antibodies targeting the gp41 N-heptad repeat (NHR),which is transiently exposed in the HIV-1 PHI,exhibit enhanced broad neutralization in cells expressing the Fc receptor,FcγRI. To enhance neutralization in cells lacking FcγRI,we here develop a bispecific antibody (bsAb) by fusing an NHR-targeting antibody to an antibody against CD4,the HIV-1 receptor on T cells. The bsAb provides a 5000-fold neutralization enhancement and shows unprecedented neutralization breadth compared to existing broadly neutralizing antibodies. Importantly,the bsAb reduces viral load in HIV-1-infected humanized male mice,and viral envelope sequencing under bsAb pressure revealed an NHR mutation that potentially impairs viral fitness. These findings validate the NHR as a potential HIV-1 therapeutic target,setting the stage for a new class of broadly neutralizing antibodies. Here the authors engineer a bispecific antibody (Ab) for HIV-1 by combining a gp41 N-heptad repeat targeting Ab with a CD4 binding Ab. Prepositioning via CD4 binding results in high neutralization breadth and potency,and experiments in HIV infected humanized male mice show reduction of viral load. View Publication