技术资料

AbstractIn this work,we have generated bispecific interleukin (IL)‐12 surrogate agonists based on camelid‐derived single‐domain antibodies (sdAbs) targeting the IL‐12 receptor (IL‐12R) subunits IL‐12Rβ1 and IL‐12Rβ2. Following immunization and antibody display‐based paratope isolation,respective sdAbs were combinatorially reformatted into a monovalent bispecific architecture by grafting resulting paratopes onto the hinge region of a heterodimeric Fc region. Functional characterization using NK‐92 cells enabled the identification of multiple different sdAb‐based bispecifics displaying divergent IL‐12R agonism capacities as analyzed by STAT4 phosphorylation. Further investigations by harnessing peripheral blood mononuclear cells (PBMCs) from healthy donors revealed attenuated pSTAT4 activation compared to recombinant human (rh) wild‐type IL‐12 regarding both natural killer (NK)‐cell and T‐cell activation but robust IL‐12R agonism on stimulated T cells. While several sdAb‐based IL‐12 mimetics were nearly inactive on NK cells as well as T cells obtained from PBMCs,they elicited significant STAT4 phosphorylation and interferon (IFN)‐γ release on stimulated T cells as well as an IL‐12‐like transcriptional signature. Furthermore,we demonstrate that the activity of receptor agonism of generated bispecific IL‐12 mimetics can also be biased towards stimulated T cells by changing the spatial orientation of the individual sdAbs within the molecular design architecture. Taken together,we present an alternative strategy to generate IL‐12‐like biologics with tailor‐made characteristics. View Publication

Post-transcriptional modifications to RNA,which comprise the epitranscriptome,play important roles in RNA metabolism,gene regulation,and human disease,including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported and investigated in the context of virus and host biology. However,the diversity of experimental approaches used has made clear correlations across studies,as well as the significance of the HIV-1 epitranscriptome in biology and disease,difficult to assess. Therefore,we established a reference HIV-1 epitranscriptome. We sequenced the model NL4–3 HIV-1 genome from infected primary CD4+ T cells and the Jurkat cell line using the latest nanopore chemistry,optimized RNA preparation methods,and the most current and readily available base-calling algorithms. A highly reproducible sense and a preliminary antisense HIV-1 epitranscriptome were created,where N6-methyladenosine (m6A),5-methylcytosine (m5C),pseudouridine (psi),inosine,and 2’-O-methyl (Nm) modifications could be identified by rapid multiplexed base-calling. We observed that sequence and neighboring modification contexts induced modification miscalling,which could be corrected with synthetic HIV-1 RNA fragments. We validated m6A modification sites with STM2457,a small molecule inhibitor of methyltransferase-like 3 (METTL3). We find that modifications are quite stable under combination antiretroviral therapy (cART) treatment,in primary CD4+ T cells,and in HIV-1 virions. Sequencing samples from people living with HIV (PLWH) revealed conservation of m6A modifications. However,analysis of spliced transcript variants suggests transcript-dependent modification levels. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor,reproducibility,and uniformity across HIV-1 epitranscriptomics studies. They also provide a roadmap for the creation of reference epitranscriptomes for many other viruses or pathogens. View Publication

The persistence of HIV-1 latency reservoirs in CD4+ T cells is a significant obstacle for curing HIV-1. Shock-and-kill strategies,which aim to reactivate latent HIV-1 followed by cytotoxic clearance,have shown limited success in vivo due to insufficient efficacy of latency reversal agents (LRAs) and off-target effects. Natural killer (NK) cells,with their ability to mediate cytotoxicity independent of antigen specificity,offer a promising avenue for enhancing the shock-and-kill approach. Previously,we observed that pan-caspase inhibitors induce NK cells to secrete an LRA in vitro. Here,we aimed to identify this LRA using a targeted proteomic approach. We identified lymphotoxin-α (LTα) as the key LRA secreted by NK cells following pan-caspase inhibitor treatment. LTα was shown to significantly induce HIV-1 LTR promoter activity,a hallmark of viral reactivation. Neutralization of LTα effectively abolished the observed LRA activity,confirming its central role. Moreover,cytokine-primed but not resting human primary NK cells exhibited LRA activity that could be neutralized with LTα neutralizing antibodies. Finally,pan-caspase inhibitor treatment did not decrease the ability of the cytokine-primed NK cells to kill target cells. These findings demonstrate that cytokine-primed NK cells,through LTα secretion,can effectively reactivate latent HIV-1 following pan-caspase inhibitor treatment,without compromising NK cell cytotoxicity. This highlights a potential enhancement strategy utilizing NK cells for shock-and-kill approaches in HIV-1 cure research. View Publication

IntroductionThe role of miRNAs in regulating variable molecular functions has been sought by scientists for its promising utility in regulating the immune response and,hence,in treating various diseases. In hepatocellular carcinoma (HCC) specifically,a reduction in the number and efficiency of circulating and intrahepatic natural killer (NK) cells has been reported. Our project aims to investigate the role of miR-216a-3p in the regulation of NK cell cytotoxicity,especially since it plays a tumor suppressor role in the context of HCC.MethodsTo achieve our aim,we isolated NK cells from the whole blood of 86 patients with HCC and 23 healthy controls. We assessed the expression profile of miR-216a-3p in NK cells of patients and controls. Furthermore,we induced the expression of miR-216a-3p in NK cells isolated from healthy controls,followed by measuring the release of interferon-gamma (IFN-γ),tumor necrosis factor-alpha (TNF-α),perforins (PRF) and granzyme B (GrB) using ELISA as well as NK cells cytolytic activity against Huh7 cells using lactate dehydrogenase (LDH) cytotoxicity assay. After that,we performed an in silico analysis to understand the mechanistic regulation imposed by miR-216a-3p on NK cells to study its impact on one of its potential downstream targets.ResultsOur results have indicated that miR-216a-3p has higher expression in NK cells of patients with HCC,and simulating this elevated expression pattern via forcing miR-216a-3p expression in normal NK cells has negatively impacted the release of TNF- α,IFN- γ,GrB,and PRF. Consequently,a decrease in cell cytolysis was observed. Our in silico analysis revealed that the predicted downstream targets of miR-216a-3p are enriched in the FOXO-signaling pathway. Among those targets is FOXO-1,which has been reported to play a role in NK cell maturation. Thus,we evaluated FOXO-1 expression upon mimicking miR-216a-3p in control NK cells that showed significant downregulation of FOXO-1 on both RNA and protein levels.ConclusionIn conclusion,we report miR-216-3p as a negative regulator of NK cell cytotoxicity. View Publication

GATA2 germline mutations lead to a syndrome characterized by immunodeficiency,vascular disorders and myeloid malignancies. To elucidate how these mutations affect hematopoietic homeostasis,we created a knock-in mouse model expressing the recurrent Gata2 R396Q missense mutation. Employing molecular and functional approaches,we investigated the mutation’s impact on hematopoiesis,revealing significant alterations in the hematopoietic stem and progenitor (HSPC) compartment in young age. These include increased LT-HSC numbers,reduced self-renewal potential,and impaired response to acute inflammatory stimuli. The mature HSPC compartment was primarily affected at the CMP sub-population level. In the mutant LT-HSC population,we identified an aberrant subpopulation strongly expressing CD150,resembling aging,but occurring prematurely. This population showed hyporesponsiveness,accumulated over time,and exhibited allele-specific expression (ASE) favoring the mutated Gata2 allele,also observed in GATA2 mutated patients. Our findings reveal the detrimental impact of a Gata2 recurrent missense mutation on the HSC compartment contributing to its functional decline. Defects in the CMP mature compartment,along with the inflammatory molecular signature,explain the loss of heterogeneity in HPC compartment observed in patients. Finally,our study provides a valuable model that recapitulates the ASE-related pathology observed in GATA2 deficiency,shedding light on the mechanisms contributing to the disease’s natural progression. View Publication

Gene enhancers often form long-range contacts with promoters,but it remains unclear if the activity of enhancers and their chromosomal contacts are mediated by the same DNA sequences and recruited factors. Here,we study the effects of expression quantitative trait loci (eQTLs) on enhancer activity and promoter contacts in primary monocytes isolated from 34 male individuals. Using eQTL-Capture Hi-C and a Bayesian approach considering both intra- and inter-individual variation,we initially detect 19 eQTLs associated with enhancer-eGene promoter contacts,most of which also associate with enhancer accessibility and activity. Capitalising on these shared effects,we devise a multi-modality Bayesian strategy,identifying 629 “trimodal QTLs” jointly associated with enhancer accessibility,eGene promoter contact,and gene expression. Causal mediation analysis and CRISPR interference reveal causal relationships between these three modalities. Many detected QTLs overlap disease susceptibility loci and influence the predicted binding of myeloid transcription factors,including SPI1,GABPB and STAT3. Additionally,a variant associated with PCK2 promoter contact directly disrupts a CTCF binding motif and impacts promoter insulation from downstream enhancers. Jointly,our findings suggest an inherent genetic coupling of enhancer activity and connectivity in gene expression control relevant to human disease and highlight the regulatory role of genetically determined chromatin boundaries. Here,the authors study the effects of expression quantitative trait loci on enhancer activity and promoter contacts in primary monocytes isolated from male individuals,suggesting an inherent genetic link between the activity of enhancers,their contacts to target gene promoters and gene expression. View Publication

Chronic lymphocytic leukemia is a malignant lymphoproliferative disorder for which primary or acquired drug resistance represents a major challenge. To investigate the underlying molecular mechanisms,we generate a mouse model of ibrutinib resistance,in which,after initial treatment response,relapse under therapy occurrs with an aggressive outgrowth of malignant cells,resembling observations in patients. A comparative analysis of exome,transcriptome and proteome of sorted leukemic murine cells during treatment and after relapse suggests alterations in the proteasome activity as a driver of ibrutinib resistance. Preclinical treatment with the irreversible proteasome inhibitor carfilzomib administered upon ibrutinib resistance prolongs survival of mice. Longitudinal proteomic analysis of ibrutinib-resistant patients identifies deregulation in protein post-translational modifications. Additionally,cells from ibrutinib-resistant patients effectively respond to several proteasome inhibitors in co-culture assays. Altogether,our results from orthogonal omics approaches identify proteasome inhibition as potentially attractive treatment for chronic lymphocytic leukemia patients resistant or refractory to ibrutinib. The molecular mechanisms underlying resistance to therapy in Chronic lymphocytic leukemia (CLL) remain to be explored. Here,the authors perform multi-omics analysis in a mouse model of ibrutinib resistance and suggest proteasome inhibition for overcoming it. View Publication

SummaryHere,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.For complete details on the use and execution of this protocol,please refer to Foltz et al.1 Graphical abstract Highlights•Protocol for inducing hESCs or iPSCs to form neural crest stem cells (NCSCs)•Steps for differentiating NCSCs into craniofacial cartilage organoids•Instructions for preparing appropriate media and conditions for differentiation•Guidance for assessing changes in cell and organoid morphology during differentiation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling. View Publication

Simple SummaryAcute myeloid leukemia (AML) is a challenging blood cancer to treat,with only about 24% of patients surviving for 5 years after diagnosis. A key challenge is that AML cells stick to normal cells in the bone marrow (BM),and these BM cells protect them from chemotherapy. The aim of this project is to find drugs that disrupt AML cell adherence to BM cells and release them into the blood,where chemotherapy will be more effective. To achieve this,we have created a model of adhesive BM and shown that it mimics the drug resistance seen clinically. We have used the model as a testing platform for drugs that disrupt AML cell adhesion. We have shown that the combined targeting of CD44 and FAK,using anti-CD44 and the clinical-grade FAK inhibitor defactinib,inhibits the adhesion of the most primitive AML cells that are associated with drug resistance and disease relapse. AbstractBackground/Objectives: Acute myeloid leukemia (AML) is an aggressive neoplasm. Although most patients respond to induction therapy,they commonly relapse due to recurrent disease in the bone marrow microenvironment (BMME). So,the disruption of the BMME,releasing tumor cells into the peripheral circulation,has therapeutic potential. Methods: Using both primary donor AML cells and cell lines,we developed an in vitro co-culture model of the AML BMME. We used this model to identify the most effective agent(s) to block AML cell adherence and reverse adhesion-mediated treatment resistance. Results: We identified that anti-CD44 treatment significantly increased the efficacy of cytarabine. However,some AML cells remained adhered,and transcriptional analysis identified focal adhesion kinase (FAK) signaling as a contributing factor; the adhered cells showed elevated FAK phosphorylation that was reduced by the FAK inhibitor,defactinib. Importantly,we demonstrated that anti-CD44 and defactinib were highly synergistic at diminishing the adhesion of the most primitive CD34high AML cells in primary autologous co-cultures. Conclusions: Taken together,we identified anti-CD44 and defactinib as a promising therapeutic combination to release AML cells from the chemoprotective AML BMME. As anti-CD44 is already available as a recombinant humanized monoclonal antibody,the combination of this agent with defactinib could be rapidly tested in AML clinical trials. View Publication

Chromosome instability is a prevalent vulnerability of cancer cells that has yet to be fully exploited therapeutically. To identify genes uniquely essential to chromosomally unstable cells,we mined the Cancer Dependency Map for genes essential in tumor cells with high levels of copy number aberrations. We identify and validate KIF18A,a mitotic kinesin,as a vulnerability of chromosomally unstable cancer cells. Knockdown of KIF18A leads to mitotic defects and reduction of tumor growth. Screening of a chemical library for inhibitors of KIF18A enzymatic activity identified a hit that was optimized to yield VLS-1272,which is orally bioavailable,potent,ATP non-competitive,microtubule-dependent,and highly selective for KIF18A versus other kinesins. Inhibition of KIF18A’s ATPase activity prevents KIF18A translocation across the mitotic spindle,resulting in chromosome congression defects,mitotic cell accumulation,and cell death. Profiling VLS-1272 across >100 cancer cell lines demonstrates that the specificity towards cancer cells with chromosome instability differentiates KIF18A inhibition from other clinically tested anti-mitotic drugs. Treatment of tumor xenografts with VLS-1272 results in mitotic defects leading to substantial,dose-dependent inhibition of tumor growth. The strong biological rationale,robust preclinical data,and optimized compound properties enable the clinical development of a KIF18A inhibitor in cancers with high chromosomal instability. Chromosomal instability occurs frequently in cancer,making it an attractive therapeutic target. Here,the authors identify KIF18A as a targetable vulnerability of cancer cells with chromosomal instability and target this using VLS-1272,a selective KIF18A inhibitor. View Publication

Cells die by necrosis due to excessive chemical or thermal stress,leading to plasma membrane rupture,release of intracellular components and severe inflammation. The clearance of necrotic cell debris is crucial for tissue recovery and injury resolution,however,the underlying mechanisms are still poorly understood,especially in vivo. This study examined the role of complement proteins in promoting clearance of necrotic cell debris by leukocytes and their influence on liver regeneration. We found that independently of the type of necrotic liver injury,either acetaminophen (APAP) overdose or thermal injury,complement proteins C1q and (i)C3b were deposited specifically on necrotic lesions via the activation of the classical pathway. Importantly,C3 deficiency led to a significant accumulation of necrotic debris and impairment of liver recovery in mice,which was attributed to decreased phagocytosis of debris by recruited neutrophils in vivo. Monocytes and macrophages also took part in debris clearance,although the necessity of C3 and CD11b was dependent on the specific type of necrotic liver injury. Using human neutrophils,we showed that absence of C3 or C1q caused a reduction in the volume of necrotic debris that is phagocytosed,indicating that complement promotes effective debris uptake in mice and humans. Moreover,internalization of opsonized debris induced the expression of pro-resolving genes in a C3-dependent manner,supporting the notion that debris clearance favors the resolution of inflammation. In summary,complement activation at injury sites is a pivotal event for necrotic debris clearance by phagocytes and determinant for efficient recovery from tissue injury. Graphical Abstract View Publication

Although chimeric antigen receptor (CAR) T cells are effective against B-lineage malignancies,post-CAR relapse is common,and efficacy in other tumors is limited. These challenges may be addressed through rational manipulations to control CAR T cell function. Here we examine the impact of cognate T cell antigen experience on subsequent CD8+ CAR T cell activity. Prior antigen encounter resulted in superior effector function against leukemia expressing low target antigen density at the expense of reduced proliferative capacity and susceptibility to dysfunction at limiting CAR doses. Distinctive temporal transcriptomic and epigenetic profiles in naive-derived and memory-derived CAR T cells identified RUNX family transcription factors as potential targets to augment the function of naive-derived CD8+ CAR T cells. RUNX2 overexpression enhanced antitumor efficacy of mouse CAR T cells,dependent on prior cell state,and heightened human CAR T cell functions. Our data demonstrate that prior antigen experience of CAR T cells determines functional attributes and amenability to transcription factor-mediated functional enhancement. Here,Fry and colleagues examine the impact of antigen experience on subsequent CD8+ CAR T cell activity during the antileukemia response and show that RUNX2 overexpression enhances antitumor activity of these cells. View Publication