技术资料

Previously,we reported that Lactobacillus paragasseri SBT2055 (LG2055) activates plasmacytoid dendritic cells (pDCs) and induces interferon alpha (IFN-α) in vitro. Our clinical trial suggested that LG2055 intake may enhance pDC activity,supporting immune maintenance and reducing subjective common cold symptoms. However,the precise mechanisms remain unclear. In this study,we investigated how LG2055 engages with pDCs to stimulate IFN-α production. We evaluated LG2055-induced pDC activation using flow cytometry,ELISA,and phagocytosis assays. Human peripheral blood mononuclear cells (PBMCs) were stimulated with LG2055 and its components to evaluate immune responses. An in vitro M cell model was used to examine LG2055 translocation. We found that DNA extracted from LG2055 activated pDCs and enhanced IFN-α production via Toll-like receptor 9 (TLR9). Phagocytosis assays demonstrated that LG2055 DNA was internalized by PBMC-derived pDCs,enabling TLR9-mediated signaling. Additionally,LG2055 translocated across M cells in vitro,suggesting potential transport into Peyer’s patches,where it may interact with pDCs. These findings demonstrate that intestinal LG2055 can translocate across M cells,interact with pDCs,and exert immune-stimulatory effects to enhance host antiviral immunity. This study provides mechanistic insight into how dietary components support immune health and could inform the development of novel functional foods or therapeutic strategies. View Publication

BackgroundT cells are contributors to atherosclerosis pathogenesis. Granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells,a specialized helper T cell subset that highly expresses GM-CSF but lacks other helper T cell markers,could exacerbate atherosclerosis development. Calycosin has been reported to suppress atherosclerosis progression. However,the effect of calycosin on ThGM cells is unknown. This study was designed to test the calycosin-induced impact on the pro-atherosclerotic function of ThGM cells in a mouse atherosclerosis model.MethodsApolipoprotein E knockout (ApoE−/−) mice were fed a high-fat diet and calycosin. The phenotype and cytokine expression of aortic ThGM cells were assessed by flow cytometry. Calycosin-derived influences on ThGM cell differentiation,proliferation,and function were determined by flow cytometry,quantitative RT-PCR,Immunoblotting,gene silencing assays,and co-culture with macrophages.ResultsAortic ThGM cell frequency was attenuated after calycosin administration. Live aortic ThGM cells,phenotypically featuring CD4+CCR6−CCR8−CXCR3−CCR10+,showed slower proliferation and weaker macrophage-activating capability in calycosin-treated mice. Besides,calycosin repressed in vitro ThGM cell differentiation and subsequently impaired ThGM cell-mediated macrophage activation,oxidized low-density lipoprotein (Ox-LDL) uptake,and foam cell formation. Importantly,calycosin upregulated nuclear receptor subfamily 4 group A member 3 (NR4A3) in ThGM cells. NR4A3 silencing partially restored the function of calycosin-treated ThGM cells.ConclusionCalycosin inhibits ThGM cell activity to suppress ThGM-cell-mediated activation of pro-atherosclerotic macrophages to ultimately ameliorate atherosclerosis progression. Therefore,we revealed a novel mechanism by which calycosin protects against atherosclerosis. View Publication

IntroductionRheumatoid arthritis (RA) is a debilitating autoimmune disease affecting approximately 1% of the global population and is associated with significant morbidity and mortality. Given the known anti-inflammatory effects of cannabinoids,we investigated the therapeutic potential of a high-CBD extract,termed CBD-X,by assessing its effects on immune cells and disease progression. This study investigates the therapeutic potential of a high-CBD extract (CBD-X) in RA.MethodsWe evaluated the effects of CBD-X on cells involved in RA pathogenesis using macrophages and primary human neutrophils as ex vivo models. In addition,two murine models of RA were applied: collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA).ResultsEx vivo experiments demonstrated that CBD-X inhibited the secretion of pro-inflammatory cytokines,including IL-1β from macrophages and IL-8,IL-6,and TNF-α from human neutrophils,suggesting its potential to modulate inflammatory responses. Moreover,CBD-X attenuated NF-κB p65 and Akt phosphorylation downstream LPS-activation signal in neutrophils. To further evaluate its therapeutic effects,we employed two murine models of RA: collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). In both models,CBD-X treatment resulted in a significant reduction of leukocyte levels in the blood,primarily through the suppression of neutrophil and monocyte populations,which play a central role in RA pathogenesis. Additionally,CBD-X reduced neutrophil migration to the joints in the CAIA model,highlighting its potential to alleviate joint inflammation. Furthermore,it modulated the neutrophil-to-macrophage ratio (NMR),an important marker of RA progression,an effect that was not observed with dexamethasone treatment,suggesting a distinct mechanism of immune regulation. Notably,CBD-X promoted the pro-resolving macrophages to the rheumatic joints. Importantly,CBD-X exerted its anti-inflammatory effect by downregulating TNF-α and MCP-1 while upregulating IL-10,a key anti-inflammatory cytokine involved in immune homeostasis.DiscussionThese findings indicate that CBD-X has a significant potential as a therapeutic agent for RA,offering a promising approach to modulate immune responses and reduce inflammation in RA patients. View Publication

IntroductionEstrogen-related receptors has been suggested as a potential therapeutic target to counteract muscle decline associated with aging or inactivity,being known to regulate mitochondrial function and cellular respiration by up-regulating key factors in muscle responses to exercise. This study aimed to evaluate the targeting of ERRs in myoblasts isolated from the skeletal muscle of inactive women by assessing the metabolic and expression changes associated with its activation.MethodsTwenty women undergoing hip arthroplasty for coxarthrosis were enrolled and divided into an active group (n = 10) and an inactive group (n = 10) based on self-reported physical activity. During surgery,muscle biopsies were taken for histological and western blotting analysis,measuring the expression levels of NADPH oxidase 4 (NOX4),sirtuin 1 (SIRT1),peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α),estrogen related receptor alpha (ERRα),and fibronectin type III domain-containing protein 5 (FNDC5). Primary cultures of myoblasts were set up from the muscle tissue of inactive women and treated with the ERRs agonist,SLU-PP-332,for subsequent qualitative and quantitative investigations. In addition,myoblasts were differentiated into myotubes for 15 days,and the success of differentiation was evaluated by immunofluorescence analysis.ResultsClinical and instrumental evaluation showed less functional limitation,higher handgrip strength values,and significantly reduced visual analogue scale scores in active subjects,in association with a significant increase in muscle fiber diameter. In addition,significantly higher expression of NOX4,concomitant with reduced levels of SIRT1,PGC-1α,ERRα,and FNDC5,was detected in the muscle tissue of inactive women. Interestingly,SLU-PP-332 treatment promoted down-regulation of NOX4 and upregulation of SIRT1,PGC-1α,ERRα,FNDC5,Akt,and B-cell lymphoma 2 (Bcl-2) in myoblasts,reducing cytotoxicity,oxidative stress,and senescence,as well as increasing levels of reduced glutathione. Furthermore,SLU-PP-332 treatment promoted abundant myotube formation,positively influencing cell differentiation.DiscussionTargeting ERRs could represent a promising therapeutic strategy to counteract muscle atrophy in elderly and sedentary subjects. However,further studies are needed to clarify the molecular mechanisms involved and explore the impact of ERRs activation on muscle metabolism. View Publication

Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success,but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion,hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet,the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable,multiplexed modulation of gene expression with greater safety. Here,we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes,PDCD1 and LAG3,both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion,limiting functional assessment,gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore,transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features. Graphical abstract Epigenome editing is used to engineer CAR T cells targeting prostate cancer by stably silencing the PDCD1 and LAG3 genes,which encode key inhibitory checkpoint receptors. This DNA break-free approach enhances safety by avoiding genomic damage and holds promise as a next-generation strategy for safer,more durable cancer immunotherapy. View Publication

Aging is associated with immune dysfunction,but long-term endurance training may confer protective effects on immune cell function. This study investigates how natural killer (NK) cell phenotypes,functional markers,and metabolism differ between endurance-trained and untrained older adults. Ex vivo expanded NK cells from endurance-trained (63.6 ± 2.1 years) and untrained (64.3 ± 3.3 years) males were exposed to adrenergic blockade (propranolol; 0–200 ng/mL) or mTOR inhibition (rapamycin; 10–100 ng/mL),both with or without PMA-induced inflammatory stimulation. Flow cytometry assessed NK subsets,activation (CD38,CD57,CD107a,NKG2D),senescence (KLRG1),and inhibitory markers (PD-1,LAG-3,TIM-3,NKG2A). Seahorse analysis measured metabolic parameters. Trained participants displayed healthier immune profiles (lower NLR,SII) and higher effector NK cells with lower cytotoxic subsets. Propranolol at 100 ng/mL blunted PMA-driven increases in CD57,CD107a,and NKG2D,while potentiating regulatory markers KLRG1,LAG-3,and PD-1 in the trained group,indicating stronger immunoregulation. With rapamycin,trained NK cells preserved NKG2D and CD107a at 10 ng/mL,maintaining cytotoxicity and degranulation. In contrast,at 100 ng/mL rapamycin plus PMA,trained NK cells shifted toward an effector phenotype with higher CD57 and CD107a,yet a blunted PMA-increased LAG-3 and TIM-3,suggesting resistance to exhaustion. PD-1 and KLRG1 remained elevated,reflecting balanced immune control. Mitochondrial analysis revealed that trained NK cells exhibited higher basal and maximal OCR,greater spare respiratory capacity,and OCR/ECAR ratio,reflecting superior metabolic fitness. These findings indicate that endurance-trained older adults have NK cells with greater functional adaptability,reduced senescence,and enhanced metabolism under inflammatory and pharmacological stress.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06057-y. View Publication

ObjectiveBifidobacterium is known to be depleted in patients with atopic dermatitis (AD). This study aims to investigate the potential prophylactic effects of Bifidobacterium animalis subsp. lactis BX-BC08 (B. lactis BX-BC08) in a murine model of AD.DesignThe immunosuppressive and anti-inflammatory effects of BX-BC08 were evaluated in a MC903-induced AD mouse model. Gut microbiota composition was analyzed by metagenomic sequencing,while high-performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to identify anti-inflammatory molecules produced by B. lactis BX-BC08.ResultsBX-BC08 significantly attenuated pro-inflammatory responses,scaling and swelling in the MC903-induced AD like murine model compared to controls. Fecal microbial profiling revealed an enrichment of probiotics and a reduction of pro-inflammatory bacteria in BX-BC08 treated mice. Metabolic analysis of BX-BC08 bacteria culture supernatant and treated mice identified a significant enrichment of alpha-Ketoglutaric acid (AKG). Functional validation in the murine AD model demonstrated that AKG strongly suppressed T helper 2 (Th2)-driven pro-inflammatory responses.ConclusionBX-BC08 mitigates AD-like inflammation by producing the anti-inflammatory metabolite AKG. BX-BC08 could serve as a novel prophylactic agent for AD prevention.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06769-9. View Publication

DEAD-box helicase 41 (DDX41) is implicated in germline (GL)-predisposed myeloid neoplasms,where pathogenic GL variants often lead to disease following the acquisition of a somatic variant in trans,most commonly p.R525H. However,the precise molecular mechanisms by which DDX41 variants contribute to the pathogenesis of myeloid neoplasms remain poorly understood,partly due to challenges in establishing cellular and animal models that faithfully recapitulate the human disease phenotype. This limitation highlights the necessity of directly analyzing primary human disease cells. In this case report,conducted to pursue this objective,we implemented single-cell RNA sequencing integrated with genotyping at the p.R525 locus in a myelodysplastic neoplasm (MDS) harboring both germline and somatic DDX41 variants,leveraging highly efficient Terminator-Assisted Solid-phase cDNA amplification and sequencing. We found that acquiring p.R525H induced G2/M cell cycle arrest selectively in colony-forming unit-erythroid cells,accompanied by R-loop accumulation,which impaired erythropoiesis through DNA damage. In hematopoietic stem and myeloid progenitor populations,gene expression profiles were largely similar between p.R525H-positive and -negative cells. However,ligand-receptor interaction and transcriptional regulation analyses suggested a non-cell-autonomous influence from p.R525H-expressing cells on GL variant-only cells. This interaction drove convergence toward a shared expression profile,highlighting an intricate interplay shaping the patient’s MDS phenotype. View Publication

AbstractBackgroundThe tumor microenvironment (TME) poses challenges that limit the efficacy of conventional CAR-T cell therapies. Homing barriers,immunosuppressive factors,and target antigen heterogeneity can impair CAR-T cell functional activity within the TME. Alternative strategies have contemplated incorporating the use of gamma delta (γδ) T cells as a CAR-T cell approach to potentially overcome these limitations. γδ T cells possess both innate and adaptive immunity to facilitate broad tumor recognition,and their natural propensity for tissue tropism may allow for more effective tumor infiltration. Reported here is the preclinical characterization of ADI-270,an allogeneic γδ CAR-T cell product targeting CD70+ cancers,engineered with a third-generation CAR based on the natural CD27 receptor. ADI-270 is also double-armored to mitigate the immunosuppressive effects of TGFβ and reduce the potential for allogeneic rejection.MethodsVδ1 T cells engineered to express an anti-CD70 CAR and dominant negative TGFβ receptor II (dnTGFβRII) were expanded from healthy donor human PBMCs. The phenotype and functional characterization of ADI-270 were assessed with in vitro cell culture assays and in vivo tumor xenograft models.ResultsADI-270 exhibited high levels of in vitro cytotoxicity against a panel of cancer cell lines and displayed a favorable inflammatory cytokine profile compared with reference scFv-based anti-CD70 CAR αβ T cells. Cytotoxicity remained potent despite low CD70 expression observed in multiple solid and hematologic tumor cell models. When armored with dnTGFβRII,ADI-270 exhibited functional resilience to TGFβ-mediated inhibition of T cell effector activity. In addition,the incorporation of potent and sensitive CD70-targeting decreased T cell-mediated alloreactive killing against ADI-270 in vitro without evidence of fratricide. Finally,ADI-270 displayed robust tumor tropism and control of primary and secondary tumor challenges in xenograft mouse models.ConclusionsThese results demonstrate the robust potency and capacity of ADI-270 to extend antitumor activity to cancers with heterogeneous antigen expression. The functional armoring incorporated into ADI-270 provides a mechanism to overcome the limitations of reduced efficacy and persistence within the TME. ADI-270 has the potential to target multiple CD70+ cancers with initial clinical evaluation proceeding in relapsed/refractory clear cell renal cell carcinoma.Trial registration numberNCT06480565. View Publication

ObjectiveB lymphocytes play a crucial role in immunity but also contribute to the pathogenesis of various diseases. Cannabis plants produce numerous biologically active compounds,including cannabinoids. The two most studied phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These cannabinoids exert diverse and potent biological effects primarily through the endocannabinoid system (ECS),which also plays a key role in mature B-cell function. Both the immune system and the ECS undergo age-related changes that lead to a clinically significant decline in function.MethodsThis study compares the effects of THC and CBD on B-cell activity in young and aged mice. Murine B lymphocytes were activated using lipopolysaccharide (LPS) and interleukin-4 (IL-4),and the impact of cannabinoid treatments was assessed in terms of cell phenotype,proliferation,antibody secretion,tumor necrosis factor-alpha (TNFα) secretion,extracellular signal-regulated kinase (ERK) phosphorylation,and the cellular metabolome.ResultsBoth THC and CBD exhibited dose-dependent inhibitory effects on B-cell activation in young and aged mice. However,we show here,for the first time,that the treatments induce distinct metabolic profiles. Although some metabolites,such as glucose-6-phosphate,pentose phosphate pathway (PPP) and nucleotide metabolites,were reduced by both cannabinoids,THC selectively reduced the levels of a distinct set of amino acids,while only CBD increased the levels of Citrulline and Allantoin. Additionally,the effects of THC and CBD differed between young and aged B cells,suggesting that age-related changes in the ECS may influence cannabinoid sensitivity.ConclusionsThese findings provide insights into the distinct mechanisms by which THC and CBD regulate immune activation and may open the door for investigating the mechanisms behind cannabinoids effects on the immune system. They also highlight the need for further research into phytocannabinoid-based therapies,particularly in age-specific contexts. Given the immunoregulatory properties of cannabinoids,especially CBD,tailored therapeutic strategies may enhance their clinical applications View Publication

BackgroundCNS stromal cells,especially fibroblasts and endothelial cells,support leukocyte accumulation through upregulation of adhesion molecules and lymphoid chemokines. While chronically activated fibroblast networks can drive pathogenic immune cell aggregates known as tertiary lymphoid structures (TLS),early stromal cell activation during CNS infection can support anti-viral T cells. However,the cell types and factors driving early stromal cell activation is poorly explored.AimsA neurotropic murine coronavirus (mCoV) infection model was used to better characterize signals that promote fibroblast networks supporting accumulation of antiviral lymphocytes. Based on the early appearance of IgD+ B cells with unknown functions during several CNS infections,we probed their potential to activate stromal cells through lymphotoxin β (LTβ),a molecule critical in maintaining fibroblast-networks in lymphoid tissues as well as promoting TLS in autoimmunity and cancers.ResultsKinetic analysis of stromal cell activation in olfactory bulbs and brains revealed that upregulation of adhesion molecules and lymphoid chemokines Ccl19,Ccl21 and Cxcl13 closely tracked viral replication. Immunohistochemistry revealed that upregulation of the fibroblast marker podoplanin (PDPN) at meningeal and perivascular sites mirrored kinetics of RNA expression. Moreover,both B cells and T cells colocalized to areas of PDPN reactivity,supporting a potential role in regulating stromal cell activation. However,specific depletion of LTβ from B cells using Mb1-creERT2 x Ltβfl/fl mice had no effect on T or B cell recruitment or viral replication. B cell depletion by anti-CD20 antibody also had no adverse effects. Surprisingly,LTβR agonism reduced viral control and parenchymal T cell localization despite increasing stromal cell lymphoid chemokines and PDPN. Additional assessment of direct stromal cell activation by the viral RNA mimic poly I:C showed induction of Pdpn and Ccl19 preceding Ltb.ConclusionsNeither B cell-derived LTβ or B cells are primary drivers of stromal cell activation networks in the CNS following mCoV infection. Although supplementary agonist mediated LTβR engagement confirmed a role for LTβ in enhancing PDPN and lymphoid chemokine expression,it impeded T cell migration to the CNS parenchyma and viral control. Our data overall indicate that stromal cells can integrate LTβR signals to tune their activation,but that LTβ is not necessarily essential and can even dysregulate protective antiviral T cell functions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03491-7. View Publication

AbstractBackgroundWhile recent clinical trials of combination immunotherapies for hepatocellular carcinoma (HCC) have shown promising clinical efficacy and survival improvements breakthroughs,there is still much room for further improvement. A key limiting factor for HCC immunotherapy is the intrinsic immunosuppression within the liver microenvironment,resulting in suboptimal priming of tumor-specific CD8 cytotoxic T cells and thus immune evasion by the tumor. Hence,identifying new key molecular pathways suppressing T-cell responses within the liver is critical for the rational design of more effective combination immunotherapies for HCC.MethodsWe identified the 5-HT2A serotonin receptor as a potential target for HCC immunotherapy in a chemical screening approach and validated that targeting 5-HT2A signaling could be a viable approach for HCC immunotherapy via in vitro and in vivo studies.ResultsDisruption of 5-HT2A signaling using either a selective antagonist small molecule,ketanserin,or by knockout of its coding gene Htr2a augments the cytotoxic effector phenotype of mouse CD8 T cells activated in vitro with immunosuppressive liver non-parenchymal cells. Ketanserin treatment of in vitro activated human CD8 T cells also increased expression of the cytotoxic effector molecules granzyme B and perforin. Abrogation of 5-HT2A signaling was associated with increased expression of cytotoxicity-related genes such as granzyme B and reduced expression of transcription factors downstream of MAP kinase signaling. In vivo,systemic ketanserin treatment significantly prolonged survival of HCC tumor-bearing mice and was non-inferior to α-programmed death ligand 1 (PD-L1)+α-vascular endothelial growth factor A (VEGFA) combination antibody treatment. Combining ketanserin with αPD-L1+αVEGFA antibodies also significantly prolonged survival relative to control-treated mice while preserving the occurrence of complete tumor regression observed with αPD-L1+αVEGFA treatment alone.ConclusionsTogether,our data describe a role for 5-HT2A as a negative regulator of the cytotoxic effector phenotype in CD8 T cells and highlight the therapeutic potential of targeting 5-HT2A for HCC immunotherapy. View Publication