技术资料

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

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

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

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

Induction of ferroptosis,an iron-dependent form of regulated cell death,holds promise as a strategy to overcome tumor resistance to conventional therapies and enhance immunotherapy responses. However,while the susceptibility of tumor cells to ferroptosis is extensively studied,limited data exists on the vulnerability of immune cells to disturbed iron balance and lipid peroxidation. Here,we found that T-cell stimulation rewires iron and redox homeostasis and by increasing levels of reactive oxygen species and labile iron promotes lipid peroxidation and T-cells’ ferroptosis. Upon stimulation,we detected changes in the balance of ferroptosis-suppressive proteins,including decrease of GPX4. Subsequently,we identified GPX4 as a master regulator orchestrating T/CAR-T-cells’ sensitivity to ferroptosis and observed that GPX4 inhibitors impair CAR-T cells’ antitumor functions. Our study demonstrated differential GPX4 expression and diverse susceptibility to ferroptosis between CD4⁺ and CD8⁺ T cells. Among analyzed subsets of naïve,central memory (CM),effector memory (EM),and terminally differentiated effector memory (TEMRA),CD8⁺ EM and CD8⁺ TEMRA cells exhibited the highest sensitivity to ferroptosis. We also showed that ferroptosis limited the anti-tumor efficacy of CAR-T cells,while ferroptosis inhibition improved their therapeutic effect,both in vitro and in vivo. Our findings are not only important to understand vulnerabilities of CAR-T cells but may also hold particular significance for their therapeutic development. In this context,future anticancer therapies should be carefully designed to selectively induce the ferroptosis of tumor cells without impeding cytotoxic cells’ antitumor efficacy. Additionally,we postulate that promoting less differentiated phenotype of CAR-T cells should be exploited therapeutically to create CAR-T products characterized by decreased sensitivity to ferroptosis within tumor microenvironment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04133-w. View Publication

High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics,yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput,low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation,allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities,demonstrating efficient delivery of various cargo,with high rates of transfection,gene knockout via non-homologous end joining,and precise knock-in through homology-directed repair. To showcase its utility in functional genomics,we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells,identifying novel regulators of exhaustion,including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-13532-z. View Publication

BackgroundDiabetic peripheral neuropathy (DPN) is one of the most prevalent and debilitating complications of diabetes,marked by chronic neuroinflammation,immune dysregulation,and progressive neuronal degeneration. Current treatments offer limited efficacy,largely focusing on symptomatic relief rather than addressing the underlying disease mechanisms. There is a critical need for disease-modifying therapies that target the molecular basis of DPN.ResultsIn this study,we developed a novel targeted nanotherapeutic system—ZH-1c-EVs@SIN—composed of neural stem cell-derived extracellular vesicles (NSC-EVs) modified with the ZH-1c aptamer and loaded with the anti-inflammatory compound sinomenine (SIN). This system was specifically designed to target microglia and inhibit the WNT5a/TRPV1 signaling pathway. Transcriptomic profiling of microglia revealed key gene networks implicated in DPN pathology and responsive to SIN treatment. Functional assays demonstrated that ZH-1c-EVs@SIN facilitated a shift in microglial phenotype from pro-inflammatory M1 to anti-inflammatory M2,significantly reduced inflammatory cytokine expression,and restored levels of neuronal regulatory proteins. Nanoparticle tracking analysis and transmission electron microscopy confirmed optimal vesicle size and morphology,while fluorescence imaging showed efficient uptake by microglia. In vivo studies in a murine model of DPN revealed marked improvements in pain-related behavior and histopathological signs of nerve damage.ConclusionZH-1c-EVs@SIN represents a promising therapeutic strategy for DPN,offering targeted immunomodulation and enhanced neural repair via regulation of the WNT5a/TRPV1 signaling axis. This nano-delivery platform introduces a novel and precise approach to intervening in diabetic neuropathy and may be applicable to other neuroinflammatory conditions.Graphical abstractMechanism of ZH-1c-EVs@SIN Mediating the WNT5a/TRPV1 Pathway to Improve Immune-Inflammatory Homeostasis in the Treatment of DPN in Mice. View Publication

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication

Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives,including thyroid,thymus,lungs,liver,and pancreas,has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage,three lineage models with two,three,and four populations and three growth models are constructed. Additionally,three models for error distribution are defined,resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration,model selection,and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184,a finding corroborated by existing literature. Additionally,space-limited growth models,such as logistic and Gompertz growth,outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model,it is predicted that the optimal differentiation period is between 1.9 and 2.4 days,plating populations closer to 300 000 cells per well result in the highest yield efficiency,and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols,providing insights into developmental biology. View Publication

The rare and rapidly progressive neurodegenerative disease multiple system atrophy (MSA) mainly affects the striatum and other subcortical brain regions. In this atypical Parkinsonian syndrome,the protein alpha-synuclein aggregates and misfolds in neurons as well as glial cells and is released in elevated amounts by hypoexcitable neurons. Mitochondrial dysregulation affects the biosynthesis of coenzyme Q10 and the activity of the respiratory chain,as shown in an induced pluripotent stem cell (iPSC) model. Proteome studies of cerebrospinal fluid and brain tissue from MSA patients yielded inconsistent results regarding possible protein changes due to small and combined groups of atypical Parkinsonian syndromes. In this study,we analysed the cellular proteome of MSA patient-derived striatal GABAergic medium spiny neurons. We observed 25 significantly upregulated and 16 significantly downregulated proteins in MSA cell lines compared to matched healthy controls. Various protein types involved in diverse molecular functions and cellular processes emphasise the multifaceted pathomechanisms of MSA. These data could contribute to the development of novel disease-modifying treatment strategies for MSA patients. View Publication

The field of human induced pluripotent stem cells (hiPSCs) has seen significant progress since the discovery of reprogramming somatic cells using the transcription factors Oct4,Sox2,Klf4,and c-Myc. hiPSCs are similar to embryonic stem cells in a primed state of pluripotency and have the potential to differentiate into any adult human cell type,offering a versatile tool for research and potential therapeutic applications. However,the efficiency of differentiation protocols for generating complex structures with multiple cell types,Like kidney organoids,remains a challenge. This study investigates the impact of treating hiPSCs with a low-dose dimethyl sulfoxide to enhance kidney organoid differentiation using the stepwise 2D monolayer-based protocol developed by Morizane et al. 2017. We found that treating hiPSCs with 1–2% DMSO affects gene expression of pluripotent transcription factors,the epigenetic landscape,and hiPSC colony morphology. Our findings also suggest DMSO treatment enhances the expression of the key metanephric mesenchyme nephron progenitor marker,SIX2 after 9 days of kidney organoid differentiation and helps improve hiPSC differentiation protocol efficiency toward the development of tubular kidney organoids. Further research is needed to fully elucidate the mechanisms underlying these effects and refine the differentiation process for potential in vitro research applications in biomedical research and drug development. View Publication

Seven female individuals with multiple congenital anomalies,developmental delay and/or intellectual disability have been found to have a genetic variant of uncertain significance in the mediator complex subunit 12 gene ( MED12 c.3412C>T,p.Arg1138Trp). The functional consequence of this genetic variant in disease is undetermined,and insight into disease mechanism is required. We identified a de novo MED12 p.Arg1138Trp variant in a female patient and compared disease phenotypes with six female individuals identified in the literature. To investigate affected biological pathways,we derived two induced pluripotent stem cell (iPSC) lines from the patient: one expressing wildtype MED12 and the other expressing the MED12 p.Arg1138Trp variant. We performed neural disease modelling,transcriptomics and protein analysis,comparing healthy and variant cells. When comparing the two cell lines,we identified altered gene expression in neural cells expressing the variant,including genes regulating RNA polymerase II activity,transcription,pre-mRNA processing,and neural development. We also noted a decrease in MED12L expression. Pathway analysis indicated temporal delays in axon development,forebrain differentiation,and neural cell specification with significant upregulation of pre-ribosome complex gene pathways. In a human neural model,expression of MED12 p.Arg1138Trp altered neural cell development and dysregulated the pre-ribosome complex providing functional evidence of disease aetiology and mechanism in MED12-related disorders. The online version contains supplementary material available at 10.1186/s10020-025-01365-5. View Publication