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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

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

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

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

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

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

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

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

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

Background/Objectives: The SARS-CoV-2’s high mutations and replication rates contribute to its high infectivity and resistance to current vaccinations and treatments. The primary cause of resistance to most current treatments aligns within the coding regions for the spike S protein of SARS-CoV-2 that has mutated. As a potential novel immunotherapy,we generated a novel fusion protein composed of a soluble ACE2 (sACE2) linked to llama-derived anti-CD16 that targets different variants of spike proteins and enhances natural killer cells to target infected cells. Methods: Here,we generated a novel sACE2-AntiCD16VHH fusion protein using a Gly4Ser linker,synthesized and cloned into the pLVX-EF1alpha-IRES-Puro vector,and further expressed in ExpiCHO-S cells and purified using Ni+NTA chromatography. Results: The fusion protein significantly blocked SARS-CoV-2 alpha,beta,delta,gamma,and omicron S-proteins binding and activating angiotensin-converting enzyme receptor-2 (ACE2) on ACE2-expressing RAW-Blue macrophage cells and the secretion of several key inflammatory cytokines,G-CSF,MIP-1A,and MCP-1,implicated in the cytokine release storm (CRS). The sACE2-Anti-CD16VHH fusion protein also bridged NK cells to ACE2-expressing human lung carcinoma A549 cells and significantly activated NK-dependent cytotoxicity. Conclusions: The findings show that a VHH directed against CD16 could be an excellent candidate to be linked to soluble ACE2 to generate a bi-specific molecule (sACE2-AntiCD16VHH) suitable for bridging effector cells and infected target cells to inhibit SARS-CoV-2 variant spike proteins binding to the ACE2 receptor in the RAW-Blue cell line and pro-inflammatory cytokines and to activate natural killer cell cytotoxicity. 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

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