技术资料

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient in vitro-derived red blood cells for clinical purposes. While BMI1,a Polycomb Repressive Complex 1 member,is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts,its mechanism of action remains poorly understood. Here we report that BMI1 overexpression leads to 10 billion-fold increase in self-renewal of human erythroblasts,which can terminally mature and agglutinate with typing reagent monoclonal antibodies. BMI1 and RING1B occupancy,along with repressive histone marks,are present at known BMI1 target genes,including the INK-ARF locus,consistent with altered cell cycle kinetics following BMI1 inhibition. Upregulation of BMI1 target genes with low repressive histone modifications,including key regulators of cholesterol homeostasis,along with functional studies,suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. We conclude that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand immature erythroid precursors for eventual clinical uses. Subject terms: Self-renewal,Cell growth,Stem-cell research View Publication

Anti-obesity medications (AOMs) have become one of the most prescribed drugs in human medicine. While AOMs are known to impact adult neurogenesis in the hypothalamus,their effects on the functional maturation of hypothalamic neurons remain unexplored. Given that AOMs target neurons in the Medial Basal Hypothalamus (MBH),which play a crucial role in regulating energy homeostasis,we hypothesized that AOMs might influence the functional maturation of these neurons,potentially rewiring the MBH. To investigate this,we exposed hypothalamic neurons derived from human induced pluripotent stem cells (hiPSCs) to Semaglutide and lipidized prolactin-releasing peptide (LiPR),two anti-obesity compounds. Contrary to our expectations,treatment with Semaglutide or LiPR during neuronal maturation did not affect the proportion of anorexigenic,Pro-opiomelanocortin-expressing (POMC+) neurons. Additionally,LiPR did not alter the morphology of POMC+ neurons or the expression of selected genes critical for the metabolism or development of anorexigenic neurons. Furthermore,LiPR did not impact the proportion of adult-generated POMC+ neurons in the mouse MBH. Taken together,these results suggest that AOMs do not influence the functional maturation of anorexigenic hypothalamic neurons. View Publication

Viral transduction is a critical step in the manufacturing of genetically modified T cells for immunotherapies,yet conventional transduction methods suffer from low to medium efficiency,high vector consumption,and limited scalability. To address these challenges,we introduce the Transduction Boosting Device (TransB),an innovative,automated,and closed-system platform designed to enable efficient and scalable gene delivery and overcome the limitations of conventional transduction methods. TransB improves cell-virus interactions by facilitating proximity between target cells and viral vectors. TransB demonstrated up to 1-fold decrease in processing time,3-fold reduction in viral vector consumption,and 0.7-fold increase in transduction efficiency compared to 24—well plate method for donor T cell transduction in studies evaluating its impact on transduction process. Comparison studies transducing T cells from three different donors with Lenti-GFP vectors showed that TransB achieved an average 0.5-fold improvement in transduction efficiencies while maintaining comparable post-transduction cell recovery,viability,growth,and phenotype compared to 24—well plate. Furthermore,TransB delivered consistent performance across two different input cell numbers demonstrating scalability of the process. These findings suggest that TransB could significantly shorten the transduction time,reduce the transduction cost and improve the transduction efficiency for manufacturing genetically modified T cell therapies. It shows strong potential as a robust,efficient,and scalable platform to enhance T cell therapy manufacturing and help overcome current manufacturing challenges in the field. The online version contains supplementary material available at 10.1186/s12967-025-06836-1. View Publication

Transplantation of engineered B cells has demonstrated efficacy in HIV disease models. B cell engineering may also be utilized for the treatment of cancer. Recent studies have highlighted that B cell activity is associated with favorable clinical outcomes in oncology. In mice,polyclonal B cells have been shown to elicit anti-cancer responses. As a potential novel cell therapy,we demonstrate that engineering B cells to target a tumor-associated antigen enhances polyclonal anti-tumor responses. We observe that engineered B cells expressing an anti-HPV B cell receptor internalize the antigen,enabling subsequent activation of oncoantigen-specific T cells. Secreted antibodies from engineered B cells form immune complexes,which are taken up by antigen-presenting cells to further promote T cell activation. Engineered B cells hold promise as novel,multi-modal cell therapies and open new avenues in solid tumor targeting. View Publication

Accumulating evidence suggests that mitogenic signaling during cell cycle arrest can lead to severe cytotoxic outcomes,such as senescence,though the underlying mechanisms remain poorly understood. Here,we explored the link between cell cycle dynamics and the formation of PML-nuclear bodies (PML-NBs),intranuclear structures known to mediate cellular stress responses. Our findings demonstrate that PML-NBs increase their number during interphase arrest. Moreover,the activation of mitogenic ERK signaling by all-trans retinoic acid (ATRA) during CDK4/6 inhibitor-induced cell cycle arrest synergistically enhances the formation of larger PML-NBs by associating with SUMO. This enlargement,triggered by the simultaneous engagement of opposing cell cycle signals,leads to potent cytotoxicity accompanied by either terminal differentiation or apoptosis,depending on the cell type,across multiple acute myeloid leukemia (AML) cell lines. Importantly,in an AML mouse model,this combination treatment significantly improved therapeutic efficacy with minimal effects on normal hematopoiesis. Our results introduce conflicting cell cycle signal-induced cytotoxicity as a promising therapeutic strategy for AML. Subject terms: PML bodies,Apoptosis View Publication

Induced pluripotent stem cells (iPSCs) are widely used to model human genetic diseases. The most common strategy involves collecting cells from relevant individuals and then reprogramming them into iPSCs. This strategy is very powerful,but finding enough individuals with a specific genetic disease can be challenging,especially since most are rare. In addition,making numerous iPSC lines is time-consuming and expensive. As a result,most studies have included relatively small numbers of iPSC lines,sometimes from the same individual. Considering the experimental variability obtained using different iPSC lines,there has been great interest in delineating the most efficient number of lines needed to achieve a robust and reproducible result. Several recommendations have been published,although most conclusions have been based on methods where experimental variance from individual cases is difficult to separate from technical issues related to the preparation of iPSCs. The current study used gene expression profiles determined by RNA sequencing (RNAseq) to empirically evaluate the impact of the number of unique individuals and the number of replicate iPSC lines from each individual for modeling Lesch-Nyhan disease (LND). This disease is caused by mutations in the HPRT1 gene,which encodes the enzyme hypoxanthine-guanine phosphoribosyltransferase. Results for detecting disease-relevant changes in gene expression depended on the analytical method employed,and whether or not statistical procedures were used to address multiple iPSC lines from the same individual. In keeping with prior studies,the best results were obtained with iPSC lines from 3-4 unique individuals per group. In contrast to prior studies,results were improved with 2 lines per individual,without statistical corrections for duplicate lines from the same individual. In the current study where all lines were produced in parallel using the same methods,most variance in gene expression came from technical factors unrelated to the individual from whom the iPSC lines were prepared. View Publication

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve,demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses,therapeutic treatments,and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic,with new subvariants continuously emerging,many harboring spike protein mutations linked to immune evasion. In this study,we characterized a panel of live SARS-CoV-2 strains,including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants,particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC),we established both monoinfections and coinfections involving SARS-CoV-2,Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably,Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020,Washington strain). Furthermore,the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead,it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6,IFN-β,and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together,our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants,as well as their interactions with other respiratory viruses,carrying important implications for therapeutic development and public health preparedness. View Publication

Immunotherapy has emerged as a key modality in cancer treatment,yet its effectiveness varies significantly among patients,often due to the metabolic stress imposed by the tumor microenvironment. Hypoxia,a major factor in the tumor microenvironment,results from the high metabolic rate of tumor cells and inadequate vascularization,impairing immune cells’ function and potentially influencing gene expression profiles. Despite the widespread use of quantitative real-time PCR in immunological studies,to the best of our knowledge,data on reference gene stability in human peripheral blood mononuclear cells under hypoxic conditions is limited. In our study,we assessed the expression stability of commonly used reference genes ( S18,HPRT,IPO8,RPL13A,SDHA,PPIA,and UBE2D2 ) in both non-stimulated and CD3/CD28-activated peripheral blood mononuclear cells cultured under normoxic,hypoxic (1% O 2 ),and chemically induced hypoxic conditions for 24 h. Analysis using four different algorithms—delta Ct,geNorm,NormFinder,and BestKeeper—identified RPL13A,S18,and SDHA as the most suitable reference genes for human peripheral blood mononuclear cells under hypoxic conditions. In contrast,IPO8 and PPIA were found to be the least suitable housekeeping genes. The study provides essential insights into the stability of reference genes in peripheral blood mononuclear cells under hypoxic conditions,a critical but understudied aspect of immunological research. Given the significant impact of hypoxia on T cell metabolism and function in the tumor microenvironment,selecting reliable reference genes is crucial for accurate gene expression analysis. Our findings will be valuable for future studies investigating hypoxia-driven metabolic reprogramming in immune cells,ultimately contributing to a better understanding of T cell responses in cancer immunotherapy. View Publication

The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed,and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites,here,we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells,PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs,and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible,concentration-dependent,and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid,propionic acid,itaconic acid,and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids,including arachidonic acid. Although lactic acid inhibited PAC,it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude,weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids. View Publication

Due to the complexity of modeling tumor-host interactions within the tumor microenvironment in vitro,we developed a 3D heterotypic cellular breast cancer (BC) model. We generated spheroid models using MCF7,MDA-MB-231,and SK-BR-3 cell lines alongside cancer-associated (BrC4f) and normal (BN120f) fibroblasts in ultra-low attachment plates. Stromal spheroids (3Df) were formed using a liquid overlay technique (graphical abstract). The YT cell line and peripheral blood NK (PB-NK) cells were used as immune components in our 3D model. In this study,we showed that stromal cells promoted tumor cell aggregation into spheroids,regardless of the initial proliferation rates,with NK cells accumulating in fibroblast-rich regions. The presence of CAFs within the model induced alterations in the expression levels of MICA/B and PD-L1 by tumor cells within the 3D-2 model. The feasibility of utilizing a 3D cell BC model in combination with cytokines and PB-NKs was evaluated. We observed that IL-15 and IL-2 enhanced NK cell activity within spheroids,whereas TGFβ had varying effects on proliferation depending on the cell type. Stimulation with IL-2 and IL-15 or TGFβ1 altered PB-NK markers and stimulated their differentiation into ILC1-like cells in 3D models. These findings underscore the regulatory function of CAFs in shaping the response of the tumor microenvironment to immunotherapeutic interventions. View Publication

HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain,HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies,independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro,HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation,cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies,HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens,in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation,cytokine secretion,and cytotoxic activity in vitro compared to either compound alone. In conclusion,HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner,without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade. View Publication

Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor family transcription factor (TF) critical for adipogenesis,lipid metabolism,insulin sensitivity,and inflammation. It has also been known to play essential roles in trophoblast development and placentation. Dysregulation of PPARG in trophoblast differentiation has been implicated in pregnancy complications,such as pre-eclampsia and gestational diabetes. However,the molecular mechanisms of PPARG-dependent target gene regulation and its interactions with other regulatory factors during human trophoblast differentiation remain unclear. Using human trophoblast stem cells (TSCs),mimicking placental cytotrophoblasts (CTs),and their differentiation into extravillous trophoblasts (EVTs) as our models,we reveal that PPARG has cell-type-specific targets in TSCs and EVTs. We also find that while PPARG is essential for both TSC self-renewal and EVT differentiation,only its role in EVT differentiation is ligand sensitive and requires ligand-binding domain (LBD)-mediated transcriptional activity,whereas its function in TSC self-renewal appears to be ligand insensitive. Combined analysis with chromosomal targets of previously defined key TFs in TSCs and EVTs shows that PPARG forms trophoblast cell-type-specific regulatory circuitries,leading to differential target gene regulation via transcriptional re-wiring during EVT differentiation. Additionally,the enhanced invasiveness of EVTs treated with a PPARG agonist suggests a potential connection between PPARG pathways and human placenta accreta. View Publication