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HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A,we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3,HAAO,HNF1A,MAP3K11) and previously unidentified (ABCD3,CDKN2AIP,USH1C,VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs,the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1,GAD2 and HOPX gene expression,potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall,our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function,and set up a framework for gene discovery and functional validation. Here,the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells,and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant. View Publication

SummaryFollicular lymphoma (FL) is an indolent non-Hodgkin lymphoma of germinal center origin,which presents with significant biologic and clinical heterogeneity. Using RNA-seq on B cells sorted from 87 FL biopsies,combined with machine-learning approaches,we identify 3 transcriptional states that divide the biological ontology of FL B cells into inflamed,proliferative,and chromatin-modifying states,with relationship to prior GC B cell phenotypes. When integrated with whole-exome sequencing and immune profiling,we find that each state was associated with a combination of mutations in chromatin modifiers,copy-number alterations to TNFAIP3,and T follicular helper cells (Tfh) cell interactions,or primarily by a microenvironment rich in activated T cells. Altogether,these data define FL B cell transcriptional states across a large cohort of patients,contribute to our understanding of FL heterogeneity at the tumor cell level,and provide a foundation for guiding therapeutic intervention. Graphical abstract Highlights•B cells from follicular lymphoma exhibit 3 distinct transcriptional states•FL B cells differ by enhanced inflammation,proliferation,or chromatin remodeling•Tumor cell states correlate with unique immune-microenvironment features•Unique mutation and CNV profiles highlight potential genetic causes of heterogeneity Krull et al. analyzed bulk transcriptional,genomic,and immune profiles of B cells from follicular lymphoma and reveal 3 distinct transcriptional states. These cell states underscore the inherent variability of FL tumors,independent of stroma,and implicate intrinsic differences as an underpinning to FL heterogeneity. View Publication

The antitumor efficacy of adoptively transferred T cells is limited by their poor persistence,in part due to exhaustion,but the underlying mechanisms and potential interventions remain underexplored. Here,we show that targeting histone demethylase LSD1 by chemical inhibitors reshapes the epigenome of in vitro activated and expanded CD8+ T cells,and potentiates their antitumor efficacy. Upon T cell receptor activation and IL-2 signaling,a timely and transient inhibition of LSD1 suffices to improve the memory phenotype of mouse CD8+ T cells,associated with a better ability to produce multiple cytokines,resist exhaustion,and persist in both antigen-dependent and -independent manners after adoptive transfer. Consequently,OT1 cells primed with LSD1 inhibitors demonstrate an enhanced antitumor effect in OVA-expressing solid tumor models implanted in female mice,both as a standalone treatment and in combination with PD-1 blockade. Moreover,priming with LSD1 inhibitors promotes polyfunctionality of human CD8+ T cells,and increases the persistence and antitumor efficacy of human CD19-CAR T cells in both leukemia and solid tumor models. Thus,pharmacological inhibition of LSD1 could be exploited to improve adoptive T cell therapy. Phenotypic changes in exhausted T cells are linked to chromatin remodeling. Here the authors show that pharmacological inhibition of the H3K4me1/2 demethylase LSD1 promotes the persistence and enhances the therapeutic activity of adoptively transferred T cells for cancer therapy. View Publication

BackgroundMacrophages play a crucial role in the development of early-onset preeclampsia (EOPE),which may be closely associated with an imbalance in macrophage M1/M2 polarization. Mesenchymal stem cell (MSC)-derived apoptotic vesicles (apoVs) have anti-inflammatory,tissue repair,and immunomodulatory functions. MSC-apoVs may ameliorate EOPE by regulating macrophage polarization,but the underlying mechanisms remain to be clarified.MethodsMacrophage infiltration and M1/M2 polarization were first analyzed in the placentas of PE patients and normal pregancies to identify macrophage alterations in EOPE placentas. MSC-apoVs were extracted and characterized. The effects of MSC-apoVs on macrophage polarization and trophoblasts invasion were validated in vivo and in vitro. miRNA transcriptomic sequencing of MSC-apoVs was conducted to identify key miRNAs involved in macrophage M2 polarization and to investigate upstream and downstream regulation factors,which were further validated in vivo and in vitro.ResultsThe proportion of M2 macrophages was significantly reduced in EOPE placentas. MSC-apoVs carrying high levels of miR-191-5p recruited macrophages,downregulated CDK6 protein expression,stabilized mitochondrial membrane potential (MMP),and promoted M2 polarization of macrophages. This enhanced the invasion of trophoblasts and improved EOPE pregnancy outcomes in mice,including reduced blood pressure,decreased urine protein,and improved embryo quality. Overexpression of miR-191-5p mimics in MSC-apoVs further alleviated EOPE-related symptoms,whereas inhibition of miR-191-5p reduced the therapeutic effect of MSC-apoVs. Further experiments confirmed that M2 macrophages polarized by MSC-apoVs promote trophoblasts invasion by secreting platelet-derived growth factor-AB (PDGF-AB),which binds to platelet-derived growth factor receptor-beta (PDGFR-β) on trophoblasts,directly activating the downstream PI3K-AKT-mTOR signaling pathway,thereby improving EOPE.ConclusionOur findings reveal the crucial role of M2 macrophages in the pathogenesis of EOPE. MSC-apoVs with high miR-191-5p recruit macrophages,downregulate CDK6,stabilize MMP,and promote M2 polarization,increasing PDGF-AB secretion,which enhances trophoblasts invasion and thereby treat EOPE. Therefore,MSC-apoVs therapy may serve as a promising strategy to improve the prognosis of EOPE.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04546-5. View Publication

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of ER,PR,and HER2 expression. Its aggressive behavior,high degree of tumor heterogeneity,and immunosuppressive tumor microenvironment (TME) are associated with poor clinical outcomes,rapid disease progression,and limited therapeutic options. Although chimeric antigen receptor (CAR)-engineered T cell therapy has shown certain promise,its applicability in TNBC is hindered by antigen escape,TME-mediated suppression,and the logistical constraints of autologous cell production. In this study,we employed hematopoietic stem and progenitor cell (HSPC) gene engineering and a feeder-free HSPC differentiation culture to generate allogeneic IL-15-enhanced,mesothelin-specific CAR-engineered invariant natural killer T ( Allo15 MCAR-NKT) cells. These cells demonstrated robust and multifaceted antitumor activity against TNBC,mediated by CAR- and NK receptor-dependent cytotoxicity,as well as selective targeting of CD1d + TME immunosuppressive cells through their TCR. In both orthotopic and metastatic TNBC xenograft models,Allo15 MCAR-NKT cells demonstrated potent antitumor activity,associated with robust effector and cytotoxic phenotypes,low exhaustion,and a favorable safety profile without inducing graft-versus-host disease. Together,these results support Allo15 MCAR-NKT cells as a next-generation,off-the-shelf immunotherapy with strong therapeutic potential for TNBC,particularly in the context of metastasis,immune evasion,and treatment resistance. The online version contains supplementary material available at 10.1186/s13045-025-01736-9. View Publication

BACKGROUND Longitudinal studies have verified the pivotal role of mesenchymal stem/stromal cells (MSCs) in the bone marrow microenvironment for hematopoiesis and coordinate contribution to leukemia pathogenesis. However,the precise characteristics and alternation of MSCs during acquired aplastic anemia (AA) remain obscure. METHODS In this study,we originally collected samples from both healthy donors (HD) and AA patients to dissect the hematological changes. To systematically evaluate the biological defects of AA-derived MSCs (AA-MSCs),we analyzed alterations in cellular morphology,immunophenotype,multi-lineage differentiation,cell migration,cellular apoptosis,and chromosome karyocyte,together with the immunosuppressive effect on the activation and differentiation of lymphocytes. With the aid of whole genome sequencing and bioinformatic analysis,we try to compare the differences between AA-MSCs and HD-derived MSCs (HD-MSCs) upon the molecular genetics,especially the immune-associated gene expression pattern. In addition,the efficacy of umbilical cord-derived MSC (UC-MSC) transplantation on AA mice was evaluated by utilizing survivorship curve,histologic sections,and blood cell analyses. RESULTS In coincidence with the current reports,AA patients showed abnormal subsets of lymphocytes and higher contents of proinflammatory cytokines. Although with similar immunophenotype and chromosome karyotype to HD-MSCs,AA-MSCs showed distinguishable morphology and multiple distinct characteristics including genetic properties. In addition,the immunosuppressive effect on lymphocytes was significantly impaired in AA-MSCs. What is more,the cardinal symptoms of AA mice were largely rescued by systemic transplantation of UC-MSCs. CONCLUSIONS Herein,we systematically investigated the signatures and efficacy of MSCs to dissect the alterations occurred in AA both at the cellular and molecular levels. Different from HD-MSCs,AA-MSCs exhibited multifaceted defects in biological characteristics and alterative molecular genetics in the whole genome. Our findings have provided systematic and overwhelming new evidence for the defects of AA-MSCs,together with effectiveness assessments of UC-MSCs on AA as well. 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

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation,development,and utilization. However,the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore,generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study,we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos,using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro,exhibiting high expression of key pluripotency markers,genomic stability,and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes. View Publication

The production of insulin-producing $\beta$ cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny,we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or $\beta$-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state,a principle that could be relevant to other systems. Notably,activation of the key $\beta$-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional $\beta$ cells. View Publication

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported,current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here,we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs.backslashnbackslashnMETHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs,an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes,a mouse cardiomyocyte cell line,but textless3% of 4 noncardiomyocyte cell types in flow cytometry analysis,which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes,which supports the specificity of MBs. Finally,MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures,and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics,which was verified by spontaneous beating,electrophysiological studies,and expression of cardiac proteins. When transplanted in a myocardial infarction model,MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.backslashnbackslashnCONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery. View Publication

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however,little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit,we demonstrate that MUC1-C is sufficient for induction of MEK → ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn,MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway,and (ii) promotes the induction of ALDH activity in breast cancer cells. View Publication