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ABSTRACTCell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors,especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response rates (ORR) have been achieved with a treatment based on CAR‐T cells in patients with malignant B‐cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR‐T cells develop high‐grade cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). However,the unprecedentedly high CRR and ORR have led to the approval of six CAR‐T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA),prompting researchers to improve existing products and develop new ones. By now,around 1000 clinical trials based on CAR‐T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases,while the remaining 16% are for solid tumors. As a result of this increased research,an enormous amount of conflicting information has been accumulated in the literature,and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information,highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR‐T cells,from manufacturing to their characterization. The focus is on all steps of the CAR‐T cell manufacturing process,from the collection of patient or donor blood to the enrichment of T cells,their activation with anti‐CD3/CD28 beads,interleukin‐2 (IL‐2) or IL‐7 and IL‐15 (induction of a more functional memory phenotype),and their transfection (viral or non‐viral methods). Automation is crucial for ensuring a standardized final product. View Publication

Co-stimulatory molecules are imperative for CD8+ T cells to eliminate target cell and maintain sustained cytotoxicity. Despite an advanced understanding of the co-stimulatory molecules deficiency that results in tumor escape,the tumor cell-intrinsic mechanisms that regulate co-stimulatory molecules remain enigmatic,and an in-depth dissection could facilitate the improvement of treatment options. To this end,in this study,we report that the deficiency of the critical costimulatory molecule CD58,mediated by the expression of ATF4 in tumor cells,impairs the formation of immunological synapses (IS) and leads to the deterioration of antitumor immune function of CD8+ T cells. Mechanistically,ATF4 transcriptionally upregulated dynamin 1 (DNM1) expression leading to DNM1-dependent endocytosis (DDE)-mediated degradation of CD58. Furthermore,administration of DDE inhibitor prochlorperazine or ATF4 knockdown effectively restored CD58 expression,boosting CD8+ T cell cytotoxicity and immunotherapy efficiency. Thus,our study reveals that ATF4 in tumor cells weakens CD58 expression to interfere with complete IS formation,and indicates potential approaches to improve the cytolytic function of CD8+ T cell in tumor immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06245-4. View Publication

Disease progression and drug resistance in patients with chronic lymphocytic leukaemia (CLL) depend on signals from the tumour microenvironment in lymphoid sites. GRK2 inhibits the egress of normal B cells from lymphoid tissues by inducing the downregulation of the S1P-receptor 1 (S1PR1). In this study we investigated the role of GRK2 in the context of CLL using in vitro and in vivo murine models,and also primary samples from CLL patients. We found that pharmacological inhibition of GRK2 enhanced the migration of leukemic cells from CLL patients towards S1P and impaired the S1P-induced downregulation of S1PR1. Likewise,CRISPR/Cas9-mediated GRK2 deletion in a murine leukemic cell line derived from the Eµ-TCL1 mouse model of CLL also increased migratory capacity toward S1P in vitro. Furthermore,when injected into mice,GRK2-deficient murine leukemic cells exhibited an altered in vivo localization,with a higher presence in the blood and spleen compared to the bone marrow. Within the spleen,these cells displayed reduced localization to the follicles compared to control murine leukemic cells. Deletion of GRK2 on murine leukemic cells did not affect their in vitro proliferation,but notably,conferred a growth disadvantage in vivo. These findings underscore GRK2 as a critical regulator of the localization of CLL cells in vivo and suggest its potential as a therapeutic target to disrupt survival niches in CLL.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-91536-5. View Publication

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways,such as treatment with nebulized instillation and inhalation. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we show the development of an mRNA therapeutic for the lower lung in mice using one-component Ionizable Amphiphilic Janus Dendrimers as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta,to produce transient protein expression in the lower regions of the lung. This study highlights a method for precise,effective,and safe delivery of TGF-β mRNA to the lung in mice. This delivery system offers a promising approach for targeting therapeutics to the specific tissues,a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. Targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease but is challenging. Here,the authors develop an mRNA delivery platform to treat acute lung injury in mice and demonstrate that it can reach the lower regions of the lung. View Publication

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

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