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BackgroundVISTA is a well-known immune checkpoint in T cell biology,but its role in innate immunity is less established. Here,we investigated the role of VISTA on anticancer macrophage immunity,with a focus on phagocytosis,macrophage polarization and concomitant T cell activation.MethodsMacrophages,differentiated from VISTA overexpressed THP-1 cells and cord blood CD34+ cell-derived monocytes,were used in phagocytosis assay using B lymphoma target cells opsonized with Rituximab. PBMC-derived macrophages were used to assess the correlation between phagocytosis and VISTA expression. qRT-PCR,flow cytometry,and enzyme-linked immunosorbent assay were performed to analyze the impact of VISTA on other checkpoints and M1/M2-like macrophage biology. Additionally,flow cytometry was used to assess the frequency of CD14+ monocytes expressing VISTA in PBMCs from 65 lymphoma patients and 37 healthy donors.ResultsEctopic expression of VISTA in the monocytic model cell line THP-1 or in primary monocytes triggered differentiation towards the macrophage lineage,with a marked increase in M2-like macrophage-related gene expression and decrease in M1-like macrophage-related gene expression. VISTA expression in THP-1 and monocyte-derived macrophages strongly downregulated expression of SIRPα,a prominent ‘don’t eat me’ signal,and augmented phagocytic activity of macrophages against cancer cells. Intriguingly,expression of VISTA’s extracellular domain alone sufficed to trigger phagocytosis in ∼ 50% of cell lines,with those cell lines also directly binding to recombinant human VISTA,indicating ligand-dependent and -independent mechanisms. Endogenous VISTA expression was predominantly higher in M2-like macrophages compared to M0- or M1-like macrophages,with a positive correlation observed between VISTA expression in M2c macrophages and their phagocytic activity. VISTA-expressing macrophages demonstrated a unique cytokine profile,characterized by reduced IL-1β and elevated IL-10 secretion. Furthermore,VISTA interacted with MHC-I and downregulated its surface expression,leading to diminished T cell activation. Notably,VISTA surface expression was identified in monocytes from all lymphoma patients but was less prevalent in healthy donors.ConclusionsCollectively,VISTA expression associates with and drives M2-like activation of macrophages with a high phagocytic capacity yet a decrease in antigen presentation capability to T cells. Therefore,VISTA is a negative immune checkpoint regulator in macrophage-mediated immune suppression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40164-024-00501-x. View Publication

Duchenne muscular dystrophy (DMD) is the most prevalent herediatry disease in men,characterized by dystrophin deficiency,progressive muscle wasting,cardiac insufficiency,and premature mortality,with no effective therapeutic options. Here,we investigated whether adenine base editing can correct pathological nonsense point mutations leading to premature stop codons in the dystrophin gene. We identified 27 causative nonsense mutations in our DMD patient cohort. Treatment with adenine base editor (ABE) could restore dystrophin expression by direct A-to-G editing of pathological nonsense mutations in cardiomyocytes generated from DMD patient-derived induced pluripotent stem cells. We also generated two humanized mouse models of DMD expressing mutation-bearing exons 23 or 30 of human dystrophin gene. Intramuscular administration of ABE,driven by ubiquitous or muscle-specific promoters could correct these nonsense mutations in vivo,albeit with higher efficiency in exon 30,restoring dystrophin expression in skeletal fibers of humanized DMD mice. Moreover,a single systemic delivery of ABE with human single guide RNA (sgRNA) could induce body-wide dystrophin expression and improve muscle function in rotarod tests of humanized DMD mice. These findings demonstrate that ABE with human sgRNAs can confer therapeutic alleviation of DMD in mice,providing a basis for development of adenine base editing therapies in monogenic diseases. Graphical abstract Li and colleagues demonstrated the efficacy of the SpG-ABE system in correcting several human nonsense mutations and restoring dystrophin expression in both patient iPSC-derived cardiomyocyte cells and humanized mouse models of DMD,indicating the potential of adenine base editing for the intervention of diseases caused by nonsense mutations. View Publication

HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically,the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition,we screened compounds specific for serine-,cysteine-,aspartyl-,and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors,including BB-94,TAPI,prinomastat,GM6001,and GI25423X,suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L,we observed association between budding HIV-1 virions and CD62L by transmission electron microscope,and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally,these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release. View Publication

As ectotherms,insects need heat-sensitive receptors to monitor environmental temperatures and facilitate thermoregulation. We show that TRPA5,a class of ankyrin transient receptor potential (TRP) channels absent in dipteran genomes,may function as insect heat receptors. In the triatomine bug Rhodnius prolixus (order: Hemiptera),a vector of Chagas disease,the channel RpTRPA5B displays a uniquely high thermosensitivity,with biophysical determinants including a large channel activation enthalpy change (72 kcal/mol),a high temperature coefficient (Q10 = 25),and in vitro temperature-induced currents from 53°C to 68°C (T0.5 = 58.6°C),similar to noxious TRPV receptors in mammals. Monomeric and tetrameric ion channel structure predictions show reliable parallels with fruit fly dTRPA1,with structural uniqueness in ankyrin repeat domains,the channel selectivity filter,and potential TRP functional modulator regions. Overall,the finding of a member of TRPA5 as a temperature-activated receptor illustrates the diversity of insect molecular heat detectors. View Publication

BackgroundRepeated exposure to sensitizing events can activate HLA-specific memory B cells,leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA).MethodsWe polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro,isolated and quantified IgG from the culture supernatant using ELISA,and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays,comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria.ResultsAt transplantation,a total of 37 DSAm were detected in 26 of 60 patients (43%),of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan–Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally,MFI levels of DSAm showed no significant association with ABMR (P=0.28).ConclusionThis study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs,particularly in non-sensitized KTRs. View Publication

SummaryThe neural network of the enteric nervous system (ENS) underlies gastrointestinal functions. However,the molecular mechanisms involved in enteric neuronal connectivity are poorly characterized. Here,we studied the role of semaphorin 5A (Sema5A),previously characterized in the central nervous system,on ENS neuronal connectivity. Sema5A is linked to autism spectrum disorder (ASD),a neurodevelopmental disorder frequently associated with gastrointestinal comorbidities,and potentially associated with ENS impairments. This study investigated in rat enteric neuron cultures and gut explants the role of Sema5A on enteric neuron connectivity and the impact of ASD-associated mutations on Sema5A activity. Our findings demonstrated that Sema5A promoted axonal complexity and reduced functional connectivity in enteric neurons. Strikingly,the ASD-associated mutation S956G in Sema5A strongly affected these activities. This study identifies a critical role of Sema5A in the ENS as a regulator of neuronal connectivity that might be compromised in ASD. Graphical abstract Highlights•Sema5A and its receptors Plexin A1/A2 are expressed by enteric neurons in rat colon•Sema5A controls synapsin 1 phosphorylation in enteric neurons•Sema5A negatively regulates neuronal activity and synaptic functions•The autism-associated mutation S956G impairs Sema5A activity Neurology; Neurogenetics View Publication

AbstractBackground and AimsFibrotic tissue formed after myocardial infarction (MI) can be as detrimental as MI itself. However,current in vitro cardiac fibrosis models fail to recapitulate the complexities of post‐MI tissue. Moreover,although MI and subsequent fibrosis is most prominent in the aged population,the field suffers from inadequate aged tissue models. Herein,an aged human post‐MI tissue model,representing the native microenvironment weeks after initial infarction,is engineered using three‐dimensional bioprinting via creation of individual bioinks to specifically mimic three distinct regions: remote,border,and scar.MethodsThe aged post‐MI tissue model is engineered through combination of gelatin methacryloyl,methacrylated hyaluronic acid,aged type I collagen,and photoinitiator at variable concentrations with different cell types,including aged human induced pluripotent stem cell‐derived cardiomyocytes,endothelial cells,cardiac fibroblasts,and cardiac myofibroblasts,by introducing a methodology which utilizes three printheads of the bioprinter to model aged myocardium. Then,using cell‐specific proteins,the cell types that comprised each region are confirmed using immunofluorescence. Next,the beating characteristics are analyzed. Finally,the engineered aged post‐MI tissue model is used as a benchtop platform to assess the therapeutic effects of stem cell‐derived extracellular vesicles on the scar region.ResultsAs a result,high viability (>74%) was observed in each region of the printed model. Constructs demonstrated functional behavior,exhibiting a beating velocity of 6.7 μm/s and a frequency of 0.3 Hz. Finally,the effectiveness of hiPSC‐EV and MSC‐EV treatment was assessed. While hiPSC‐EV treatment showed no significant changes,MSC‐EV treatment notably increased cardiomyocyte beating velocity,frequency,and confluency,suggesting a regenerative potential.ConclusionIn conclusion,we envision that our approach of modeling post‐MI aged myocardium utilizing three printheads of the bioprinter may be utilized for various applications in aged cardiac microenvironment modeling and testing novel therapeutics. View Publication

SummaryOvarian clear cell carcinoma (OCCC) is a gynecological cancer with a dismal prognosis; however,the mechanism underlying OCCC chemoresistance is not well understood. To explore the intracellular networks associated with the chemoresistance,we analyze surgical specimens by performing integrative analyses that combine single-cell analyses and spatial transcriptomics. We find that a chemoresistant OCCC subpopulation with elevated HIF activity localizes mainly in areas populated by cancer-associated fibroblasts (CAFs) with a myofibroblastic phenotype,which is corroborated by quantitative immunostaining. CAF-enhanced chemoresistance and HIF-1α induction are recapitulated in co-culture assays,which show that cancer-derived platelet-derived growth factor (PDGF) contributes to the chemoresistance and HIF-1α induction via PDGF receptor signaling in CAFs. Ripretinib is identified as an effective receptor tyrosine kinase inhibitor against CAF survival. In the co-culture system and xenograft tumors,ripretinib prevents CAF survival and suppresses OCCC proliferation in the presence of carboplatin,indicating that combination of conventional chemotherapy and CAF-targeted agents is effective against OCCC. Graphical abstract Highlights•HIF-1α-positive cancer cells and CAFs form chemoresistant niches of OCCC•PDGF from HIF-1α-positive cancer cells activates CAFs•Activated CAFs induce HIF-1α expression and chemoresistance in cancer cells•Inhibition of PDGF signaling in CAFs augments chemosensitivity of cancer cells Through an integration of spatial analyses and co-culture studies,Mori et al. demonstrate that feedback activation between HIF-1α-positive cancer cells and cancer-associated fibroblasts (CAF) contributes to chemoresistance of ovarian clear cell carcinoma. Targeted inhibition of CAF activation by a receptor tyrosine kinase inhibitor augmented chemosensitivity,suggesting possible application for CAF-targeted therapy. View Publication

BackgroundMyxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene,however,the precise functional contributions of these two elements remain enigmatic in tumor development.MethodsTo study the cell-free microenvironment in MLS,we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently,scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing.ResultsCharacterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions,as well as chromatin remodeling,immune response,and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS.ConclusionsOur experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05211-w. View Publication

SUMMARY The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here,we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically,Sel1L is required for optimal bioenergetics and c-Myc expression. Finally,we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together,these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence. Graphical Abstract In brief Correa-Medero et al. report that CD8+ T cells undergo dynamic ER stress during an acute viral infection,and they demonstrate a cell-intrinsic role for Sel1L,the ER-associated degradation adaptor,in CD8+ T cell effector function,persistence,and optimal cellular metabolism. View Publication

During brain development,neural progenitors expand through symmetric divisions before giving rise to differentiating cell types via asymmetric divisions. Transition between those modes varies among individual neural stem cells,resulting in clones of different sizes. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyse clonal behaviour of entire tissues are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids,to show that individual stem cell clones produce progeny on a vastly variable scale. By using stochastic modelling we find that variable lineage sizes arise because a subpopulation of lineages retains symmetrically dividing cells. We show that lineage sizes can adjust to tissue demands after growth perturbation via chemical ablation or genetic restriction of a subset of cells in chimeric organoids. Our data suggest that adaptive plasticity of stem cell populations ensures robustness of development in human brain organoids. Lindenhofer,Haendeler,Esk,Littleboy et al. perform whole-tissue lineage tracing in human cerebral organoids to reveal that a subpopulation of symmetrically dividing cells can adjust its lineage size depending on tissue demands. View Publication

AbstractThe mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy,with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses,resistance to chemotherapy,and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9,which is associated with a poor prognosis,increased chemoresistance,and higher relapse rates. However,direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA,leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors,namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models,DB1055 and DB818 induced in vitro cell growth reduction,death,differentiation,and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore,DB1055 and DB818 exhibited potent antileukemic activities in a human THP‐1 AML in vivo model,leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients,with DB1055 successfully reducing leukemia burden in patient‐derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9. View Publication