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SummarymRNA-based in vivo chimeric antigen receptor (CAR)-T cell engineering offers advantages over ex vivo therapies,including streamlined manufacturing and transient expression. However,current delivery methods require antibody-modified vehicles with manufacturing challenges. In this study,inspired by cardiolipin,we identify cardiolipin-like di-phosphoramide lipids that improve T cell transfection without targeting ligands,both in vitro and in vivo. The T cell-favored tropism is likely due to the lipid’s packing,shape,and rigidity. Encapsulating circular RNA further prolongs mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles,we deliver mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR),alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single-cell sequencing in humans confirms uPAR’s relevance to senescence and inflammation in RA. To facilitate clinical translation,we screen and humanize single-chain variable fragments (scFvs) against uPAR,establishing a PL40 mRNA-encoded humanized uPAR CAR with potential for treating aging-inflamed disorders. Graphical abstract Highlights•Cardiolipin-mimic phosphoramide (CAMP) LNPs transfect T cells without antibody modification•Circular mRNA prolongs mRNA expression•Senolytic in vivo CAR-T treats inflamm-aging disease (liver fibrosis and rheumatoid arthritis)•Develop humanized anti-human uPAR scFv Zhang et al. develop Cardiolipin-mimic phosphoramide (CAMP) lipids,which enable T cell transfection without antibody modification. Using CAMP-based LNPs,they generate senolytic CAR-T cells in vivo to target inflamm-aging diseases. Additionally,they employ circular mRNA to prolong transgene expression. The authors also engineer a humanized anti-human uPAR scFv for clinically relevant applications. View Publication

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2),a receptor expressed on microglia,is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques,and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore,TREM2 could serve as a non-invasive biomarker for AD. In this study,we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474,0.807,and 0.415 ng/mL,respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis. View Publication

AbstractBackgroundThe pro-inflammatory cytokine,interleukin-18 (IL-18),plays an instrumental role in bolstering anti-tumor immunity. However,the therapeutic application of IL-18 has been limited due to its susceptibility to neutralization by IL-18 binding protein (IL-18BP),short in vivo half-life,and unfavorable physicochemical properties.MethodsIn order to overcome the poor drug-like properties of IL-18,we installed an artificial disulfide bond,removed the native,unpaired cysteines,and fused the stabilized cytokine to an IgG Fc domain. The stability,potency,pharmacokinetic and pharmacodynamic properties as well as efficacy of disulfide-stabilized IL-18 Fc-fusion (dsIL-18-Fc) were assessed via in vitro and in vivo studies.ResultsThe stability and mammalian host cell production yields of dsIL-18-Fc were improved,compared to the wild-type (WT) cytokine,while maintaining its biological potency and interactions with IL-18 receptor α (IL-18Rα) and IL-18BP. Recombinant fusion of the cytokine to an IgG Fc domain provided extended half-life. Notably,despite maintaining sensitivity to IL-18BP,dsIL-18-Fc was effective at activating both T and natural killer (NK) cells,and elicited a strong anti-tumor response,either as a single agent,or in conjunction with anti-programmed cell death-ligand 1 (anti-PD-L1) therapy.ConclusionsWe engineered IL-18 for reinforced stability,extended half-life,and improved manufacturability. The therapeutic benefit of dsIL-18-Fc,coupled with a more favorable manufacturability profile and enhanced drug-like properties,underscores the potential utility of this engineered cytokine in cancer immunotherapy. View Publication

Menopause not only affects fertility but also has widespread impact on systemic health. Yet,the molecular mechanisms underlying this process are not fully understood,partly due to the absence of robust,age-relevant preclinical models with comprehensive molecular and phenotypic characterization. To address this,we systematically compared three candidate mouse models of menopause: (1) intact aging,(2) chemical ovarian follicle depletion using 4-vinylcyclohexene diepoxide (VCD) administered at multiple ages,and (3) Foxl2 haploinsufficiency,a genetic model based on a transcription factor linked to human premature ovarian failure. Through histology,serum hormone profiling,single-cell transcriptomics and machine-learning approaches,we uncovered both shared and model-specific features of follicle loss,endocrine disruption,and transcriptional remodeling. The VCD and Foxl2 haploinsufficiency models revealed distinct patterns of hormonal and immune alterations not captured by intact aging alone. This comparative framework enables informed selection of context-appropriate preclinical rodent models to study menopause and the broader physiological consequences of ovarian aging. View Publication

Imaging macrophage trafficking in solid tumors has major implications for cancer diagnosis,prognosis,and therapy. Here,we show that macrophage labeling with lipid-shelled microbubbles enables ultrasound imaging at single-cell level. Crucially,microbubble labeling and sonication at low mechanical indexes do not affect macrophage viability,migration,phenotype,and cytokine secretion profile,supporting the notion that ultrasound imaging can be used for nondestructive macrophage imaging. Despite the damping exerted on the microbubble oscillations by the cellular compartments,the microbubbles exhibit highly nonlinear behavior upon sonication,allowing for high specificity nonlinear US imaging under in vitro and in vivo conditions. Subsequently,we demonstrate that nonlinear ultrasound imaging can selectively monitor macrophage accumulation and extravasation in solid tumors in rodents for at least 8 h after intravenous administration. These findings establish ultrasound as a noninvasive platform for immune cell trafficking in solid tumors and highlight its potential to advance cancer diagnosis,monitoring,and therapy. Imaging macrophage trafficking in solid tumors has implications for cancer diagnosis and prognosis. by labeling macrophages with lipid-shelled microbubbles in combination with ultrasound,the authors here achieve nondestructive in vivo intravenously administrated macrophage imaging at single cell level with 100 µm resolution till 8 h in solid tumors in rodents. View Publication

Cytoplasmic stress granules (SG) assemble in response to stress-induced translational arrest and are key signaling hubs orchestrating cell fate and regulating various physiological and pathological processes. However,the role of SG formation in the progression of allergic diseases is incompletely understood. Here,by analyzing the nasal tissues of allergic rhinitis (AR) mouse models and AR patients,we find that SGs assemble specifically in the macrophages within the nasal mucosa and promote AR progression by restraining the efferocytotic ability of macrophages,ultimately resulting in reduced Mres generation and IL-10 production. Mechanistically,intracellular m7G-modified Lrp1 mRNA,encoding for a typical efferocytosis receptor,is transported by the m7G reader QKI7 into stress-induced SGs,where Lrp1 mRNA is sequestered away from the translation machinery,ultimately resulting in reduced macrophage efferocytosis. Therefore,SG assembly impairs macrophage efferocytosis and aggravates AR,and the inhibition of SGs bears considerable potential in the targeted therapy. Cytoplasmic stress granules (SG) regulate cell fate and are involved in several physiological and pathological processes. Here,using mouse models of allergic rhinitis (AR),the authors reveal the formation of SGs within macrophages of the nasal mucosa and implicate SGs in the regulation of Lrp1-mediated efferocytosis and Type 2 cytokine production,aggravating AR symptoms. View Publication

CD8+ T cell exhaustion (Tex) limits immune control of cancer,but the underlying molecular drivers are unclear. In the present study,we identified the prostaglandin I2 (prostacyclin) receptor PTGIR as a cell-intrinsic regulator of T cell exhaustion. Transcriptomic profiling of terminally exhausted (Ttex) CD8+ T cells revealed increased activation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response pathway. Enhancing NRF2 activity (by conditional deletion of Kelch-like ECH-associated protein 1 (KEAP1)) boosts glutathione production in CD8+ T cells but accelerates terminal exhaustion. NRF2 upregulates PTGIR expression in CD8+ T cells. Silencing PTGIR expression enhances T cell effector function (that is,interferon-γ and granzyme production) and limits Ttex cell development in chronic infection and cancer models. Mechanistically,PTGIR signaling impairs T cell metabolism and cytokine production while inducing transcriptional features of Tex cells. These findings identify PTGIR as a NRF2-dependent immune checkpoint that regulates balance between effector and exhausted CD8+ T cell states. Targeting CD8+ T cell exhaustion is a strategy to enhance immune checkpoint inhibition and to fight cancer. Here the authors show a NRF2-dependent role for the prostaglandin I2 receptor PTGIR in controlling T cell exhaustion. View Publication

Background: Virotherapy eradicates tumors by directly killing cancer cells and causing adjuvant effects. However,the mechanism by which non-replicating virotherapy exerts anti-tumor effects is unclear. Methods: In this study,we investigated the genes that mediate the anti-tumor effects of ultraviolet (UV)-irradiated Hemagglutinating Virus of Japan envelope (HVJ-E) using RNA sequencing,gene knockout,and a drug-inducible gene expression system. We examined the antitumor effects of Apolipoprotein d (Apod) using genome-wide CRISPR library screening,in situ biotinylation combined with mass spectrometry,flow cytometry,biochemistry,and tumor-bearing mouse models. Results: Here,we show that HVJ-E represses tumor growth via Irf7-induced Apod expression in tumor cells in vivo. Irf7 in B16F10 cells is a pivotal transcription factor for HVJ-E-induced anti-tumor effects. Apod substantially suppresses tumor growth even in HVJ-E-insensitive tumors. Apod is required to increase NKG2D-ligand genes in HVJ-E-treated tumors. Genome-wide CRISPR library screening and in situ biotinylation of Apod reveal an association of Apod with ERK2. Mechanistically,Apod prevents the nuclear translocation of ERK2 and Importin7,increasing NKG2D-ligands expression in B16F10 cells and attenuating tumor growth. Treating a local tumor with a combination therapy of Apod with the anti-OX40,T cell costimulatory molecule,antibody substantially repressed tumor growth in target and non-target lesions alongside T cell activation. Conclusion: Our findings provide insights into the molecular mechanisms of how HVJ-E induces anti-tumor effects and can aid the development of therapeutic strategies for eliciting anti-tumor immunity. View Publication

Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people and decreased iron availability impairs immunity. Nevertheless,how iron deprivation impacts immune cell function remains poorly characterised. We interrogate how physiologically low iron availability affects CD8+ T cell metabolism and function,using multi-omic and metabolic labelling approaches. Iron limitation does not substantially alter initial post-activation increases in cell size and CD25 upregulation. However,low iron profoundly stalls proliferation (without influencing cell viability),alters histone methylation status,gene expression,and disrupts mitochondrial membrane potential. Glucose and glutamine metabolism in the TCA cycle is limited and partially reverses to a reductive trajectory. Previous studies identified mitochondria-derived aspartate as crucial for proliferation of transformed cells. Despite aberrant TCA cycling,aspartate is increased in stalled iron deficient CD8+ T cells but is not utilised for nucleotide synthesis,likely due to trapping within depolarised mitochondria. Exogenous aspartate markedly rescues expansion and some functions of severely iron-deficient CD8+ T cells. Overall,iron scarcity creates a mitochondrial-located metabolic bottleneck,which is bypassed by supplying inhibited biochemical processes with aspartate. These findings reveal molecular consequences of iron deficiency for CD8+ T cell function,providing mechanistic insight into the basis for immune impairment during iron deficiency. Iron has been shown to be necessary for the activation and differentiation of CD8+ T cells. Here the authors investigate changes in CD8+ T cell metabolism in iron limiting conditions and find that aspartate is increased yet downstream nucleotide synthesis is suppressed and addition of exogenous aspartate partially rescues T cell function. View Publication

Despite the success of chimeric antigen receptor T (CART) cell therapy in hematological malignancies,durable remissions remain low. Here,we report CART senescence as a potential resistance mechanism in 41BB-costimulated CART cell therapy. To mimic cancer relapse,we utilized an in vitro model with repeated CART cell activation cycles followed by rest periods. Using CD19-targeted CART cells with costimulation via 4-1BB-CD3ζ (BBζ) or CD28-CD3ζ (28ζ),we showed that CART cells undergo functional,phenotypical,and transcriptomic changes of senescence,which is more prominent in BBζ. We then utilized two additional independent strategies to induce senescence through MYC activation and irradiation. Induction of senescence impaired BBζ activity but improved 28ζ activity in preclinical studies. These findings were supported by analyses of independent patient data sets; senescence signatures in CART cell products were associated with non-response to BBζ but with improved clinical outcomes in 28ζ treatment. In summary,our study identifies senescence as a potential mechanism of failure predominantly in 41BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. SignificanceWe identified senescence as a cause of failure in CART cell therapy,predominantly in 4-1BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. View Publication