技术资料

CD8+ T cells are central to targeting and eliminating cancer cells. Their function is critically supported by type 1 conventional dendritic cells (cDC1s),which both prime antigen‐specific CD8+ T cells in tumour‐draining lymph nodes (tdLNs) and sustain primed CD8+ T cells within tumours. Despite their importance,the spatiotemporal organisation of cDC1s within tumours and their diverse functional roles remain poorly understood. Here,we use scRNAseq and unbiased spatial analysis to construct a detailed map of cDC1 states and distribution within immunogenic mouse tumours during CD8+ T‐cell‐mediated rejection. We reveal two distinct cDC1 activation states characterised by differential expression of genes linked to anti‐tumour immunity,including Cxcl9 and Il12b. Strikingly,Il12b‐expressing cDC1s are CCR7+ and enriched at tumour borders,where they closely associate with stem‐like TCF1+ CD8+ T cells. In contrast,CCR7–Cxcl9‐expressing cDC1s are preferentially found within the tumour parenchyma alongside effector CD8+ T cells. Analysis of a published dataset of human tumours similarly reveals a spatial association between CCR7+ cDC1 and stem‐like TCF1+ CD8+ T cells. These findings uncover a highly spatially coordinated interaction between cDC1s and CD8+ T cells within tumours,shedding light on the intricate cellular dynamics that underpin effective anti‐tumour immunity. Using scRNAseq and spatial analysis,we analyse cDC1 states and spatial distribution in tumours during immune‐mediated rejection. We identify two cDC1 activation states,each occupying different regions and associated with distinct CD8+ T cell populations. This reveals the spatial organisation of cDC1 states that may be key to anti‐tumour immunity. View Publication

IntroductionT cells are major components of the immune system. Their activation requires interaction between the T cell receptor and co-stimulatory molecules,crucial during infection,inflammation,and allogeneic rejection. Monomeric CRP (mCRP) is a known modulator of inflammation and particularly the innate immune response,however its interaction with T cells as part of the adaptive immune response remains unclear.MethodsPeripheral blood mononuclear cells (PBMC) and T cells were isolated. Flow cytometric analysis was conducted to evaluate Fcγ receptor CD16 expression on T cells,the binding of CRP to T cells,and its impact on proliferation and apoptosis. T cell activation was assessed after 1,2,3,5 and 7 days by assessing CD69 and CD25 expression,and under various conditions including coculture with monocytes and several inhibitory factors.ResultsT cells express CD16 that binds mCRP in a concentration-dependent manner,and particularly on activated T cells. While mCRP reduces apoptosis and accelerates proliferation in T cells,it does not independently activate them. However,activation of monocytes by mCRP leads to T cell activation,indicating a direct cell to cell interaction during CRP-induced activation. This effect could be alleviated by inhibition of the CD80/CD28 pathway.ConclusionCRP does not activate T Cells directly but via PI3-kinase-dependent activation of monocytes and subsequent CD80/CD28 cell to cell contact. The findings suggest the effects of CRP on T cells depend on their environment and the presence of other proinflammatory agents. View Publication

FoxP3+ regulatory T (Treg) cells restrict excessive immune responses and immunopathology as well as reactivity to self or environmental antigens and thus are crucial for peripheral immune tolerance. The transcription factor Interferon Regulatory Factor 4 (IRF4) controls differentiation and function of T cells. In Treg cells,IRF4 is required for peripheral activation and maturation to effector Treg (eTreg) cells with enhanced suppressive function. However,the mechanisms of Treg cell regulation by IRF4 are not fully understood. Here,we analyze the role of IRF4 in differentiation and maintenance of Treg cells using IRF4-deficient mice and a T cell transfer model,that allows Irf4 inactivation in peripheral T cells. We demonstrate that loss of one Irf4 allele already results in impaired eTreg cell differentiation and decreased Treg cell homeostasis,indicating that IRF4 controls peripheral Treg cell differentiation in a gene dosage dependent mode. Peripheral Irf4 inactivation was also associated with enhanced production of inflammatory but also inhibitory cytokines by Treg cells. ATAC sequencing of Treg cells after mutation of one or both Irf4 alleles revealed regions with altered accessibility in genes involved in Treg cell function. In the FoxP3 gene,Irf4 inactivation resulted in reduced ATAC signals in the promoter region and in the conserved non-coding sequence (CNS) 2,required for stability of FoxP3 expression in peripheral Treg cells in response to TCR stimulation. IRF4-deficient Treg cells also displayed a reduction in open chromatin in several Treg cell specific super enhancers,mainly located in proximity to potential IRF4 binding sites. In conclusion,our results demonstrate that IRF4 controls peripheral Treg cell differentiation and homeostasis in a gene dosage dependent manner. View Publication

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

SummaryA strong interest in drugs targeting the tumor microenvironment (TME) necessitates new experimental systems that incorporate key TME components. Compared to traditional 2D cell lines,3D ex vivo spheroids from patient-derived xenograft (PDX) materials may better capture patient tumor characteristics. We developed and validated a 3D tumor spheroid model from non-small cell lung cancer (NSCLC) PDXs to enable T cell infiltration. Histologic and transcriptomic analysis suggested that tumor spheroids closely recapitulate the source PDX tumor tissues. Consistent T cell infiltration into tumor spheroids was achieved using a well-established magnetic nanoparticle technology,which maintained T cell function and tumor-killing activity. Drug treatment studies with immunotherapy agents also demonstrated the potential scalability of 3D tumor-T cell spheroids in assessing drug activity,including tumor viability and cytokine secretion. This platform provides a useful tool for evaluating drug candidates that can be translated to patient tumor responses related to both tumor intrinsic and TME factors. Graphical abstract Highlights•We developed a 3D tumor spheroid model from lung cancer patient-derived xenografts•The model enabled robust T cell infiltration and preserved T cell cytotoxic functions•Histology and RNA-seq showed that tumor spheroids closely resembled source tumors•Proof-of-concept experiments showed this platform’s utility in preclinical drug testing Biological sciences; Biotechnology; Natural sciences; Tissue Engineering View Publication

BackgroundAlthough both pre-clinical and clinical studies show promising outcomes,resulting in rapid growth of clinical trials of MSC-based therapies in recent years,the heterogeneity and therapeutic inconsistency of MSCs have severely hampered their clinical applications. Purifying homogenous MSC populations with enhanced specific functions represents one promising approach. We have demonstrated recently that the CD317+ MSCs have enhanced anti-inflammatory functions and improved therapeutic efficacy and consistency.MethodsIn the current study,we performed both in vitro and in vivo investigations to delineate whether and how CD317 regulates the immune modulation function of MSCs.ResultsOur data here indicate that the CD317 directly contributes to the immune suppression function of MSCs stimulated by TNF-α through up-regulating TSG6 via CD317/lipid-raft/TNFR1 complex. The CD317 stabilizes the TNFR1 complex,resulting in hyper-activation of the NF-κB pathway and up-regulation of TSG6,which confers the therapeutic effects of MSCs on the mouse model of ALI (acute lung injury) and IBD (inflammatory bowel disease).ConclusionsThus,the CD317 stabilizes TNFR1 and confers the anti-inflammatory functions of MSCs via NF-κB/TSG6 Pathway.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04527-8. 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

The nucleocapsid N is one of four structural proteins of the coronaviruses. Its essential role in genome encapsidation makes it a critical therapeutic target for COVID-19 and related diseases. However,the inherent disorder of full-length N hampers its structural analysis. Here,we describe a stepwise method using viral-derived RNAs to stabilize SARS-CoV-2 N for EM analysis. We identify pieces of RNA from the SARS-CoV-2 genome that promote the formation of structurally homogeneous N dimers,intermediates of assembly,and filamentous capsid-like structures. Building on these results,we engineer a symmetric RNA to stabilize N protein dimers,the building block of high-order assemblies,for EM studies. We combine domain-specific monoclonal antibodies against N with chemical cross-linking mass spectrometry to validate the spatial arrangement of the N domains within the dimer. Additionally,our cryo-EM analysis reveals novel antigenic sites on the N protein. Our findings provide insights into N protein´s architectural and antigenic principles,which can guide design of pan-coronavirus therapeutics. The authors stabilize the SARS-CoV-2 nucleocapsid (N) dimer assembly using a short RNA and chemical crosslinker for EM analysis,revealing its domain arrangement and antigenic sites to advance understanding and guide pan-coronavirus therapeutic design. View Publication

Anti-melanoma differentiation-associated gene 5 (MDA5)-positive dermatomyositis (DM) is often complicated by rapidly progressive interstitial lung disease (RP-ILD),leading to early mortality. Previous studies on the pathogenesis of anti-MDA5-positive DM highlighted type I interferons (IFNs),while recent investigations reported the significance of a type III IFN,IFN-λ3. We investigated a range of cytokines,including type I/II/III IFNs,in serum samples from anti-MDA5-positive DM patients collected at diagnosis before treatment introduction. Elevations of IFN-β and λ3 were identified as the hallmark of anti-MDA5-positive DM,in comparison with other myositis subtypes,systemic lupus erythematosus,and COVID-19 pneumonia. The elevation of IFN-λ3 was associated with decreased CD56dimCD16pos NK cells in circulation. The unique cytokine profile with type I/III IFN upregulation in anti-MDA5-positive DM was replicated in independent validation cohorts. A cluster analysis using serum type I/III IFN levels identified three subgroups in anti-MDA5-positive DM: mild elevations of IFN-α/β and λ3; a marked increase in IFN-λ3 alone; and pronounced elevations of IFN-α/β with mild to moderate increase in IFN-λ3. Patients in the cluster with a marked elevation of IFN-λ3 alone tended to present with RP-ILD and decreased survival. The combination of serum type I/III IFN levels could serve as a prognostic biomarker in anti-MDA5-positive DM. View Publication

IntroductionCD8+ T cells are vital in the immune control of cancer and a key player in cell-based cancer immunotherapy. Recent studies have shown that microbial short-chain fatty acids (SCFA) can promote both effector and memory phenotypes in CD8+ T cells and may thereby enhance protection against cancer.MethodsIn this study,we determined the effect of SCFA butyrate on mouse CD8+ T cell function in vitro and in vivo,using the OT-I model.ResultsButyrate co-culture with anti-CD3 + anti-CD28 activated T cells in vitro enhanced the frequency of effector CD8+ IFN-γ-producing cells,and the amount of cytokine produced per cell. Culture with butyrate also enhanced the activation,TCR expression,and levels of phosphorylated mTOR proteins within CD8+ T cells but reduced proliferation rate and increased apoptosis. Butyrate-treated activated cells conferred tumor protection after adoptive transfer. Butyrate-treated cells were present at higher frequencies within the tumor compared to non-butyrate treated cells,and expressed IFN-γ. When analyzed using high dimensional cytometry,the tumors of mice that received butyrate-treated cells were enriched in clusters displaying an effector memory phenotype with high expression of IL-15Rβ and T-bet.DiscussionOur findings show that butyrate promotes the effector activity of CD8+ T cells in culture,which can persist in vivo while also stimulating memory phenotypes. Consequently,butyrate treatment may have strong application in T cell-based immunotherapies to improve protective cell functions and patient outcomes. View Publication