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HIV infection remains incurable as the virus persists within a latent reservoir of CD4+T cells. Novel approaches to enhance immune responses against HIV are essential for effective control and potential cure of the infection. In this study,we designed a novel tetravalent bispecific antibody (Bi-Ab32/16) to simultaneously target the gp120 viral protein on infected cells,and the CD16a receptor on NK cells. In vitro,Bi-Ab32/16 triggered a potent,specific,and polyfunctional NK-dependent response against HIV-infected cells. Moreover,addition of the Bi-Ab32/16 significantly reduced the latent HIV reservoir after viral reactivation and mediated the clearance of cells harboring intact proviruses in samples from people with HIV (PWH). However,the in vivo preclinical evaluation of Bi-Ab32/16 in humanized mice expressing IL-15 (NSG-Hu-IL-15) revealed a significant decline of NK cells associated with poor virological control after ART interruption. Our study underscores the need to carefully evaluating strategies for sustained NK cell stimulation during ART withdrawal. Bispecific antibody targeting NK cells facilitates clearance of HIV-infected cells in vitro but poses challenges in sustaining NK cell function during ART withdrawal in preclinical models. View Publication

AbstractBackgroundChimeric antigen receptor (CAR) therapies have demonstrated potent efficacy in treating B-cell malignancies,but have yet to meaningfully translate to solid tumors. Nonetheless,they are of particular interest for the treatment of glioblastoma,which is an aggressive form of brain cancer with few effective therapeutic options,due to their ability to cross the highly selective blood-brain barrier.MethodsHere,we use our pooled screening platform,CARPOOL,to expedite the discovery of CARs with antitumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×106 third generation CARs targeting IL-13Rα2,a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity,proliferation,memory formation,and persistence on repeated antigen challenge.ResultsEach enriched CAR robustly produced the phenotype for which it was selected,and one enriched CAR triggered potent cytotoxicity and long-term proliferation on in vitro tumor rechallenge. It also showed significantly improved persistence and comparable tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma,but also demonstrated increased off-target recognition of IL-13Rα1.ConclusionTaken together,this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date. View Publication

Waldenstrom’s Macroglobulinemia (WM) is an IgM-secreting bone marrow (BM) lymphoma that is preceded by an asymptomatic state (AWM). To dissect tumor-intrinsic and immune mechanisms of progression,we perform single-cell RNA-sequencing on 294,206 BM tumor and immune cells from 30 patients with AWM/WM,26 patients with Smoldering Myeloma,and 23 healthy donors. Despite their early stage,patients with AWM present extensive immune dysregulation,including in normal B cells,with disease-specific immune hallmarks. Patient T and NK cells show systemic hypo-responsiveness to interferon,which improves with interferon administration and may represent a therapeutic vulnerability. MYD88-mutant tumors show transcriptional heterogeneity,which can be distilled in a molecular classification,including a DUSP22/CD9-positive subtype,and progression signatures which differentiate IgM MGUS from overt WM and can help advance WM research and clinical practice. The impact of tumor intrinsic and immune alterations on disease progression in patients with Waldenstrom’s Macroglobulinemia (WM) remains to be characterized. Here,the authors perform single-cell RNA-sequencing and identify distinct tumor subtypes,tumour microenvironment features and potential therapeutic vulnerabilities in patients with WM. 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

How macrophages in the tissue environment integrate multiple stimuli depends on the genetic background of the host,but this is still poorly understood. We investigate IL-4 activation of male C57BL/6 and BALB/c strain specific in vivo tissue-resident macrophages (TRMs) from the peritoneal cavity. C57BL/6 TRMs are more transcriptionally responsive to IL-4 stimulation,with induced genes associated with more super enhancers,induced enhancers,and topologically associating domains (TAD) boundaries. IL-4-directed epigenomic remodeling reveals C57BL/6 specific enrichment of NF-κB,IRF,and STAT motifs. Additionally,IL-4-activated C57BL/6 TRMs demonstrate an augmented synergistic response upon in vitro lipopolysaccharide (LPS) exposure,despite naïve BALB/c TRMs displaying a more robust transcriptional response to LPS. Single-cell RNA sequencing (scRNA-seq) analysis of mixed bone marrow chimeras indicates that transcriptional differences and synergy are cell intrinsic within the same tissue environment. Hence,genetic variation alters IL-4-induced cell intrinsic epigenetic reprogramming resulting in strain specific synergistic responses to LPS exposure. Genetic background affects how macrophages integrate multiple stimuli,e.g.,to IL-4 in tissue environments. BALB/c macrophages show different transcriptional and epigenomic remodeling compared to C57BL/6,leading to distinct synergistic LPS responses. 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

BackgroundNeutrophil extracellular trap (NET) formation has been implicated as a pathogenic mechanism in both rheumatoid arthritis (RA) and interstitial lung disease (ILD). However,the role of NETs in RA-associated ILD (RA-ILD) and the mechanisms driving NET formation remain unclear. This study aimed to assess the involvement of NETs in RA-ILD and elucidate the underlying mechanisms.MethodsSingle-cell sequencing was used to identify changes in the quantity and function of neutrophils in the lung tissue of a zymosan A (ZYM)-induced interstitial pneumonia arthritis model. Additionally,nuclear receptor 4A3 (NR4A3) interference was performed in HL-60 cells to assess its impact on NET formation and the transformation of MRC-5 cells into myofibroblasts. The clinical relevance of plasma myeloperoxidase-DNA (MPO-DNA),citrullinated histone 3 (Cit-H3),and cell-free DNA was evaluated in RA-ILD patients with different imaging types via a commercial enzyme-linked immunosorbent assay (ELISA).ResultsIn the ZYM-treated SKG mouse model,which recapitulates key features of RA-ILD,an increased population of neutrophils in the lung tissue was primarily responsible for NET formation. Mechanistically,we found that interference with NR4A3 expression enhanced NET formation in HL-60 cells,which in turn promoted the differentiation of MRC-5 cells into myofibroblasts. Clinically,plasma MPO-DNA levels are elevated in patients with RA-nonspecific interstitial pneumonia (RA-NSIP),whereas Cit-H3 levels are elevated in RA-usual interstitial pneumonia (RA-UIP) patients compared with healthy subjects. ROC curve analysis further revealed that the combination of plasma MPO-DNA,rheumatoid factor (RF),and anti-citrullinated protein (anti-CCP) and the combination of Cit-H3,RF,and anti-CCP were superior diagnostic panels for NSIP and UIP in RA-ILD patients,respectively. Moreover,compared with those from healthy controls,neutrophils from patients with RA-UIP and RA-NSIP demonstrated a significantly increased ability to form NETs and induce the differentiation of MRC-5 cells into myofibroblasts. Specifically,RA-UIP patients exhibited a greater capacity for NET formation and the differentiation of MRC-5 cells into myofibroblasts than did RA-NSIP patients.ConclusionsThese findings suggest that targeting NETs may be a novel therapeutic approach for treating ILD in RA patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12931-025-03111-1. 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

Donor-specific antibodies (DSAs) targeting mismatched human leukocyte antigen (HLA) molecules are one of the principal threats to long-term graft survival in solid organ transplantation. However,many patients with long-term circulating DSAs do not manifest rejection responses,suggesting a degree of heterogeneity in their pathogenicity and related functional activity. Immunologic risk stratification of transplant recipients is complicated by challenges intrinsic to defining alloantibody responses that are potentially pathogenic versus those that are not. Thus,a comprehensive understanding of how human alloantibodies target and interact with donor HLA molecules is vital for the development and evaluation of new strategies aimed at reducing antibody-mediated rejection responses. In this study,we employ hydrogen–deuterium exchange–mass spectrometry (HDX–MS),molecular dynamics (MD) simulations,and advanced biochemical and biophysical methodologies to thoroughly characterize a panel of human monoclonal alloantibodies and define the influence of Fc-region biology,antibody binding kinetics,target antigen density,and structural characteristics on their ability to potentiate the forms of immune effector mechanisms that are strongly implicated in transplant rejection. Our findings have significant implications for our understanding of the key biological determinants that underlie the pathogenicity or lack thereof of human alloantibodies. View Publication