技术资料

Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations. AbstractFc engineering to enhance antibody effector functions harbors the potential to improve therapeutic effects. Understanding FcγR expression and distribution in the tumor microenvironment prior to and following treatment may help guide immune-engaging antibody design and patient stratification. In this study,we investigated FcR-expressing immune effector cells in HER2+ and triple-negative breast cancers (TNBC),including neoadjuvant chemotherapy–resistant disease. FcγRIIIa expression,FcγRIIIa+ NK cells,and classically activated (M1-like) macrophages correlated with improved anti-HER2 antibody efficacy. FcγRIIIa protein and FcγRIIIa+ NK cells and macrophages were present in primary TNBC and retained in treatment-resistant tumors. FcγRIIIa was spatially associated with folate receptor alpha–positive (FRα+) tumor areas at baseline and in residual tumors following neoadjuvant chemotherapy. Wild-type and Fc-engineered antibodies recognizing two breast cancer–associated antigens,HER2 and the emerging TNBC target FRα,were designed and generated to have increased FcγRIIIa-expressing effector cell engagement. The combination of glycoengineering,including fucose removal from the N-linked Fc glycan,and Fc point mutations greatly increased antibody affinity for and retention on FcγRIIIa. The Fc-engineered antibodies enhanced immune effector activity against HER2+ breast cancer and TNBC,altering proinflammatory cytokine production by NK cells and tumor-conditioned macrophages and skewing macrophages toward proinflammatory states. Furthermore,the Fc-engineered antibodies restricted orthotopic HER2+ and FRα+ breast cancer xenograft growth at doses suboptimal for equivalent wild-type antibodies and recruited FcγRIIIa-expressing cells into tumors. Antibody design through combined glycoengineering and Fc point mutations to enhance FcγRIIIa engagement of tumor-infiltrating effector cells may be a promising strategy for developing therapies for patients with aggressive and treatment-resistant breast cancers.Significance:Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations. View Publication

Ataxia with oculomotor apraxia type 1 (AOA1) is a rare,autosomal recessive,early-onset,progressive cerebellar ataxia caused by mutations in the APTX gene,which encodes aprataxin,a DNA-adenylate hydrolase involved in DNA damage repair. The pathogenesis of AOA1 remains unclear. The purpose of this study was to investigate the pathogenesis of a novel mutation,p.H201P/H201R,carried by our AOA1 patient and the mechanism of AOA1 in an induced pluripotent stem cells (iPSCs) model. We edited iPSCs derived from a healthy individual to carry the APTX homozygous mutation p.H201P (H201P-iPSCs) or p.H201R (H201R-iPSCs) via CRISPR/Cas9. We found that aprataxin expression was absent in both H201P- and H201R-iPSCs. The capacity of these APTX-mutant iPSCs to differentiate into neural progenitor cells (NPCs) and mature neurons was diminished. We observed an increase in DNA single-strand breaks (SSB) via a comet assay and poly(ADP-ribose) staining,and an increase in the ratio of cleaved PARP-1/total PARP-1 in APTX-mutant NPCs and early immature neurons (EiNs),in addition of a heightened sensitivity to tert-butyl hydroperoxide in APTX-mutant EiNs. Moreover,a decrease of APE1 expression was observed in APTX-mutant NPCs and H201R-EiNs during neural differentiation. Our study established a practical iPSCs model to investigate AOA1 disease. We found that mutant aprataxin leads to defective neural differentiation,accompanied by the accumulation of DNA SSBs with increased cleaved PARP-1 and reduced APE1 expression of the base excision repair pathway. View Publication

Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD),dexamethasone,IL‐10,or rapamycin. However,challenges remain in obtaining robust and efficient generation of cell therapy‐based tolDCs without compromising their viability. We recently reported that CCR2‐dependent recruitment of monocytic cells,with the capacity to dampen T‐helper responses,occurs in mice treated with a single‐stranded oligonucleotide (ssON). Here,we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL‐4 and GM‐CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD‐L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes,many of which overlapped with VitD‐moDC and resulted in moDCs with altered lipopolysaccharide (LPS)‐responsiveness. Moreover,ssON‐moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4+FoxP3+CD25+ T cells. Experiments using chemical reagents support a role for PPAR‐γ in the generation of ssON‐moDC. Collectively,our data show that monocytes differentiated with IL‐4,GM‐CSF,and ssON generate cells with phenotypic and functional characteristics of tolDCs. In this article,the authors elucidated the immunoregulatory role of an oligonucleotide (ssON) that favors the induction of human tolerogenic dendritic cells (DC). The tolerogenic profile was evidenced by reduced responsiveness to lipopolysaccharides (LPS) (A). Importantly,the tolerogenic DCs had upregulated PD‐L1 molecules and functionally inhibited the proliferation of alloreactive T cells and induced FoxP3+ Tregs (B). This study envisions the development of ssON as therapeutic for rebalancing overactive T‐helper cell responses. View Publication

Aggressive non‐Hodgkin lymphoma (aNHL) often relapses after first‐line treatment. Clinical data supports the safety and efficacy of the combination of mosunetuzumab,a CD20×CD3 bispecific antibody,and polatuzumab vedotin,an anti‐CD79b antibody drug conjugate (Mosun‐Pola) in relapsed/refractory aNHL. This study investigated the molecular mechanism behind the combination effect of Mosun‐Pola in human diffuse large B‐cell lymphoma (DLBCL) cell lines. Methods: The in vitro Mosun‐Pola efficacy in DLBCL cells (SU‐DHL‐8 and HT) was evaluated by T cell‐dependent cellular cytotoxicity (TDCC) assay. CD20‐stable‐knockdown SU‐DHL‐8 cells were established using lentiviral short hairpin RNA. Surface and T‐cell activation marker proteins expression were determined by flow cytometry. Human T‐cell‐injected mice or humanized NOD/Shi‐scid,IL‐2Rγnull (huNOG) mice were used for an in vivo study. Results: An in vitro TDCC assay showed a synergistic effect in SU‐DHL‐8 and HT cells. Based on our experimental results of suppressing CD20 expression,it was suggested that this combination effect could be caused by an increase in CD20 expression by polatuzumab vedotin. In addition,examining the effects of CD20 upregulation in tumor cells on T‐cell activation demonstrated that the combination of Mosun‐Pola enhanced T‐cell activation markers in both CD4+ and CD8+ T cells during the TDCC reaction. In vivo studies,using human immune system‐reconstituted mouse models confirmed that polatuzumab vedotin enhanced CD20 expression in tumors,and the combination of Mosun‐Pola showed significantly improved anti‐tumor effects compared with single‐drug treatments. Conclusions: These findings suggest that polatuzumab vedotin‐induced CD20 upregulation provides a molecular rationale to explain the synergistic effect of this combination therapy.Trial RegistrationThe authors have confirmed clinical trial registration is not needed for this submission. View Publication

Alzheimer’s disease (AD) involves cognitive decline,amyloid-beta (Aβ) accumulation,tau hyperphosphorylation,and neuroinflammation. CREB1,a key transcription factor for memory,is downregulated in AD,contributing to disease progression. Phosphodiesterases 4 and 10 (PDE4 and PDE10) are key enzymes that degrade cAMP,a second messenger involved in CREB signaling,synaptic plasticity,and neuroprotection. Dysregulation of PDE activity has been implicated in AD and other neurodegenerative disorders. Methods: We used human iPSC-derived cortical neurons and microglia,along with the APP/PS1 mouse model,to investigate the role of CREB1 and assess the therapeutic potential of dual PDE4/10A inhibition in AD. Results: CREB1 deficiency in neurons increased Aβ and p-tau231 accumulation. Dual inhibition of PDE4 and PDE10A activated the cAMP-PKA-CREB pathway,restoring CREB1 activity,reducing Aβ and p-tau231,and mitigating neuroinflammation. This intervention improved synaptic plasticity and cognitive performance in vivo. Conclusions: Our findings demonstrate that dual PDE4/10A inhibition synergistically enhances the cAMP-PKA-CREB signaling,promoting neuroprotection and synaptic remodeling. This approach offers a promising therapeutic strategy for modifying AD pathology and restoring cognitive function. View Publication

Bispecific T cell-engagers (BTEs) are engineered antibodies that redirect T cells to target antigen-expressing tumors. BTEs targeting tumor-specific antigens such as interleukin 13 receptor alpha 2 (IL13RA2) and epidermal growth factor receptor variant III (EGFRvIII) have been developed for glioblastoma (GBM). However,there is limited mechanistic understanding of the action of BTE since prior studies were mostly conducted in immunocompromised animal models. To close this gap,the function of BTEs was assessed in the immunosuppressive tumor microenvironment (TME) of orthotopic and genetically engineered mouse models (GEMM) with intact immune systems. Method: sA BTE that bridges CD3 epsilon on murine T cells to IL13RA2-positive GBM cells was developed,and the therapeutic mechanism was investigated in immunocompetent mouse models of GBM. Multicolor flow cytometry,single-cell RNA sequencing (scRNA-seq),multiplex immunofluorescence,and multiparametric MRI across multiple preclinical models of GBM were used to evaluate the mechanism of action and response. Results: BTE-mediated interactions between murine T cells and GBM cells triggered T cell activation and antigen-dependent killing of GBM cells. BTE treatment significantly extended the survival of mice bearing IL13RA2-expressing orthotopic glioma and de novo forming GBM in the GEMM. Quantified parametric MRI validated the survival data,showing a reduction in glioma volume and decreased glioma viability. Flow cytometric and scRNA-seq analyses of the TME revealed robust increases in activated and memory T cells and decreases in immunosuppressive myeloid cells within the brains of mice following BTE treatment. Conclusions: Our data demonstrate that the survival benefits of BTEs in preclinical models of glioma are due to the ability to engage the host immune system in direct killing,induction of immunological memory,and modulation of the TME. These findings provide a deeper insight into the mechanism of BTE actions in GBM. View Publication

Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13,resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus,activation of these silenced genes can serve as a therapeutic target for PWS. Here,we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably,these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally,the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders. The authors develop CRISPR-based epigenome editing strategy to reactivate silenced maternally inherited genes for Prader-Willi syndrome in human iPSC and hypothalamic organoid models,highlighting its potential for treating imprinting disorders. View Publication

We used high-resolution optical mapping (~50 µm) to investigate potential arrhythmia mechanisms following transplantation of engineered cardiac tissue. We induced myocardial infarction in 6 immunosuppressed pigs and implanted cardiac spheroids into the border zone. One week later,600-µm-thick cardiac slices containing implanted spheroids were harvested and electrical propagation was imaged. Histology showed low connexin-43 expression,scar,and misaligned muscle fibers at the graft-host interface. We observed propagation from host-to-graft in 10 slices from 3 pigs. Host-graft electrical bridges were spaced by millimeters. Propagation was ~4-fold slower in the graft than host. One graft beat spontaneously,but activation did not propagate from graft-to-host in this,or any other slice. We did not observe reentry,but slow in-graft conduction and sparse electrical bridges provided opportunity for reentry induction. These data reveal potential for reentrant or focal arrhythmias 1 week post-implant,which may resolve with maturation of the graft and the graft-host interface. View Publication

Phosphatidylinositol 3-kinase delta (PI3KCD) is a critical signaling enzyme for B cell development,activation,function and immune regulation. Gain-of-function mutations in PI3KCD result in the congenital immunodeficiency known as Activated PI3KCD Syndrome (APDS). APDS patients are prone to repeated infections and other serious clinical manifestations. Here,we determine how B cell-intrinsic expression of the APDS-associated PI3KCDE1021K mutation impacts immune responses to the protozoan parasite Trypanosoma congolense. PI3KCDE1021K/B mice exhibit a significant expansion of IL10-expressing B cells within the spleen and peritoneal cavity,which was associated with impaired control of T. congolense infection. Despite the generation of robust germinal center,plasma cell and antibody responses,PI3KCDE1021K/B mice show elevation in the first wave of parasitemia and increased mortality. We further characterize the phenotype of the expanded IL10-producing B cell population in PI3KCDE1021K/B mice,which show hallmarks of innate-like regulatory B cells (Breg) and expression of multiple inhibitory molecules. This Breg expansion is associated with reduced IFNγ/IL10 ratio,reduced TNFα production and impaired activation of myeloid cells,likely compromising the innate response to infection. These findings highlight the profound impact of dysregulated PI3KCD activity on regulatory B cells that can functionally impair innate immune responses controlling a systemic parasite protozoan disease. Author summaryB cells and antibodies play a critical role in the immune response to Trypanosome parasites. Molecular signaling networks within B cells can control the type of response generated during infection. Here,we studied how a genetic variant in the signaling enzyme PI3KCD,previously linked to human immune deficiencies,impacts B cell responses to Trypanosome infection. We find that mice expressing the PI3KCDE1021K mutation in their B cells show impaired control of Trypanosome infection,and alterations in several aspects of the immune response. Specifically,we noted these mice poorly control parasite growth within the first week of infection,a timeframe where specific antibody responses have not yet been generated. We noted an altered balance between pro-inflammatory and anti-inflammatory cytokine mediators produced within the first week of infection. This was associated with high numbers of regulatory B cells expressing multiple molecules capable of inhibiting other cells of the immune system. We further found that these mice show functional alterations in other critical immune cell types,such as macrophages and T cells. These findings highlight the impact of dysregulated PI3KCD activity on regulatory B cells that can impair immune responses controlling a systemic parasite protozoan disease. View Publication

Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation. But the mechanisms underlying this primed pluripotent state,which endows them with elevated responsiveness to differentiation cues,remain largely underexplored. Especially,little is known about the pivotal transcription factors (TFs) that orchestrate hESCs towards primed pluripotency. Here,we report a function of TF ZNF263 in pluripotency priming. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs,greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly,ZNF263 deficiency markedly impairs pluripotency dissolution and multi-lineage differentiation in hESCs,particularly toward ectoderm. Moreover,single-cell transcriptomic profiling reveals that ZNF263 promotes the priming of cell fate commitment in hESCs,suggesting its indispensable requirement for pluripotency priming and lineage commitment continuum. Together,we demonstrate the role of ZNF263 in establishing the primed pluripotent state in hESCs and facilitating their differentiation into primary germ layer lineages. Human embryonic stem cells are simultaneously capable of self-renewal and poised for differentiation. Here,the authors show a role for the ZNF263 transcription factor promotes primed pluripotency and facilitates differentiation into primary germ layer lineages. View Publication

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here,we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation,integrating gene expression profiling,label-free quantitative proteomics,high-resolution respirometry,fluorescence lifetime imaging microscopy (FLIM),and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation,including enhanced oxidative phosphorylation,increased mitochondrial content,and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways,while FLIM indicated a progressive increase in enzyme-bound NAD(P)H,consistent with a shift toward oxidative metabolism. Notably,13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites,together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs,indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function,differentiated neurons maintained glycolytic activity,suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease. View Publication

Cancer stem cells were prominent responsible for cancer initiation,metastasis,and invasion as well as therapeutic resistance in colorectal cancer (CRC). The extracellular axon guidance factor netrin-1 has been found to be overexpressed in several malignant cancers such as glioma,lung cancers,and colorectal cancer. However,the role of netrin-1 on cancer stemness in CRC remains unveiled. Our study revealed high expression of netrin-1 in colorectal cancer tissues and its ability to promote cancer stemness by interacting with receptors UNC5B and neogenin on murine colorectal cancer cell. Mechanistically,the netrin-1-UNC5B/neogenin axis activates the downstream NF-κB and ERK1/2 signaling pathways,reinforcing the stemness properties of tumor cells,and further exacerbating tumor progression. Clinically,netrin-1 expression associated with poor survival and high CD133 expression in patients with CRC. Taken together,these results suggest that netrin-1 blockade could be a compelling therapeutic strategy to improve the poor outcomes and trigger cancer stemness inhibition in CRC treatment. View Publication