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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.