技术资料

Drug resistance and relapse are still major challenges in acute myeloid leukemia (AML) because of the inability to effectively eradicate leukemia stem cells (LSCs). Senescence induction combined with immune killing may offer promising strategies for LSC eradication. However,whether and how drug-resistant LSCs retain stemness via senescence and immune regulation remains unknown. The immunoproteasome subunit PSMB10 expression levels were analyzed by single-cell RNA-seq data,along with the bioinformatics analysis of publicly available AML datasets,and quantified using RT-qPCR and flow cytometry (FCM) analysis on clinical samples from AML patients. The cellular senescence was evaluated by the assays of cell proliferation,cell cycle,senescence-associated β-galactosidase activity,and senescence-associated secretory phenotype factors. In vitro T-cell killing assay was played to determine immune escape reprogramming of AML cells. FCM was conducted to estimate intracellular drug concentration and cellular apoptosis rates. Human AML xenografts and PSMB10 knockout syngeneic mouse bone marrow transplantation models were utilized to investigate the function of PSMB10. Various techniques were employed for mechanism studies,including Lentivirus transduction or siRNA transfection,western blotting,co-immunoprecipitation assays,luciferase reporter assays,polysome profiling assays,quantitative proteomics,etc. PSMB10 mRNA was significantly upregulated in the surviving nonsenescent LSCs,exhibiting a 13-fold increase compared to senescent LSCs following chemotherapy. The specific high expression of PSMB10 in post-chemotherapy nonsenescent LSCs predicts a poor AML prognosis. The genetic inactivation of PSMB10 resulted in increased senescence and cytotoxic T lymphocyte (CTL) killing,as well as increased intracellular drug concentrations and drug-induced cellular senescence in different types of human AML cells,which also impeded human and murine leukemia initiation and stemness maintenance in vivo with a 19-fold decrease in the frequency of human LSCs and a 7.6-fold decrease of drug-resistant mouse LSCs,while normal hematopoietic cells remained unaffected. Mechanistically,the downregulation of PSMB10 boosted SLC22A16-mediated drug endocytosis and further induced chemotherapy drug-mediated senescence through the RPL6/RPS6-MDM2-P21 pathway in AML cells. Additionally,downregulating PSMB10 also impeded MHC-I protein degradation-induced escape of CTL killing. PSMB10 is a key candidate molecular target for eradicating drug-resistant LSCs via senescence and immune regulation. The online version contains supplementary material available at 10.1186/s13046-025-03420-9. View Publication

Testicular somatic cells play an important role in supporting spermatogenesis. Leydig cells (LCs) and peritubular myoid cells (PTMs) originate from a common progenitor population and show similar expression signatures in adulthood,making it difficult to distinguish and isolate the two in vitro. In this study,new surface markers for identifying adult LCs (ALCs) and PTMs were discovered by reanalyzing testicular single-cell dataset. Differential expressions of ITGA9 and NGFR were confirmed through immunofluorescence staining of human testes. A novel Fluorescence activated Cell Sorting (FACS) protocol is established for the isolation of ALCs and PTMs based on the two markers. Long-term culture of both cells were performed and their characteristics were characterized and explored. ITGA9+ /NGFR + cells were positive for markers of PTMs (SMA,CNN1) and negative for markers of ALCs (HSD3B,STAR),and were able to form tubular and spheroid structures in vitro. In contrast,ITGA9-/NGFR + cells were positive for ALC markers and negative for PTM markers,and showed a capacity of testosterone production in vitro. Also,both cells were negative for Sertoli cell marker SOX9. When the two cells were cultured,they can expand for more than 15 passages. Our study established a novel and efficient method for identifying and isolating human ALCs and PTMs,which provides a great potential for researches of the two cell types in human. The online version contains supplementary material available at 10.1186/s12958-025-01389-w. View Publication

Dynamic regulation of metabolic activities in astrocytes is critical to meeting the demands of other brain cells. During neuronal stress,lipids are transferred from neurons to astrocytes,where they are stored in lipid droplets (LDs). However,it is not clear whether and how neuron-derived lipids trigger metabolic adaptation in astrocytes. Here,we uncover an endolysosomal function that mediates neuron-astrocyte transcellular lipid signaling. We identify Tweety homolog 1 (TTYH1) as an astrocyte-enriched endolysosomal protein that facilitates autophagic flux and LD degradation. Astrocyte-specific deletion of mouse Ttyh1 and loss of its Drosophila ortholog lead to brain accumulation of neutral lipids. Computational and experimental evidence suggests that TTYH1 mediates endolysosomal clearance of ceramide 1-phosphate (C1P),a sphingolipid that dampens autophagic flux and LD breakdown in mouse and human astrocytes. Furthermore,neuronal C1P secretion induced by inflammatory cytokine interleukin-1β causes TTYH1-dependent autophagic flux and LD adaptations in astrocytes. These findings reveal a neuron-initiated signaling paradigm that culminates in the regulation of catabolic activities in astrocytes. Subject terms: Organelles,Glial biology,Lipid signalling View Publication

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here,we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45,CD56,CD14,and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells,as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis,which also revealed colocalization of lymphoid markers with carbonic anhydrase 9,a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells by tumor cells. Finally,we show evidence of chromosomal DNA being transferred from immune cells to tumor cells through physical contact. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell,resulting in a fusion phenotype that expresses both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally,further studies into trogocytosis and other mechanisms of contact-mediated cellular transfer will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other cancers. View Publication

This study investigates the impact of minimal residual disease (MRD) on relapse in patients with acute myeloid leukemia (AML),focusing on its interaction with immune cells function. A total of 49 AML patients were enrolled in this prospective study and categorized into four groups: MRD − positive with relapse,MRD − positive without relapse,MRD − negative with relapse,and MRD − negative without relapse. Peripheral blood T lymphocyte subpopulations were analyzed using ten-color flow cytometry. CD4 + T cells were co-cultured with leukemia cell lines to assess the impact of CD4 + T cells on leukemia cell proliferation,apoptosis,and cytokine release. In MRD − positive patients,relapsed individuals exhibited significantly higher levels of CD4 + T cells,regulatory T (Treg) cells,and CD4 + CD45RA + naïve T cells compared to non-relapsed patients ( P < 0.0001,P = 0.0016,and P = 0.0066,respectively). Conversely,in MRD − negative patients,relapsed individuals showed a significantly lower percentage of Treg cells ( P = 0.0068). Furthermore,we observed that CD4 + T cells were associated with enhanced leukemia cell proliferation and reduced apoptosis,along with markedly increased IL-10 expression. The available data raise the possibility that CD4 + T cell-derived IL-10 participates in immune microenvironment regulation,a process that may have implications for MRD maintenance and disease recurrence in AML. View Publication

The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here,we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo,thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies,we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes,resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin,we show resistance of engrafted,multiplex edited human cells in vivo,and a 2-fold enrichment for edited cells in vitro. Together,our results highlight the potential of adenine base editors for improved immune and gene therapies. Subject terms: Haematopoietic stem cells,Bone marrow transplantation,Cell biology View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication

Bats can host viruses of pandemic concern without developing disease. The mechanisms underlying their exceptional resilience to viral infections are largely unresolved,necessitating the development of physiologically relevant and genetically tractable research models. Here,we developed respiratory and intestinal organoids that recapitulated the cellular diversity of the in vivo epithelium present in Rousettus aegyptiacus,the natural reservoir for the highly pathogenic Marburg virus (MARV). In contrast to human counterparts,bat organoids and mucosal tissue exhibited elevated constitutive expression of innate immune effectors,including type I interferon-ε (IFNε) and IFN-stimulated genes (ISGs). Upon infection with diverse zoonotic viruses,including MARV,bat organoids strongly induced type I and III IFN responses,which conferred robust antiviral protection. Type III IFNλ3 additionally displayed virus-independent self-amplification,acting as an ISG to enhance antiviral immunity. Our organoid platform reveals key features of bat epithelial antiviral immunity that may inform therapeutic strategies for viral disease resilience. Subject terms: Mucosal immunology,Viral infection View Publication

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation,survival and therapy resistance of acute myeloid leukemia (AML) cells. Here,we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA),a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD + . We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis,glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML,we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry,and ROS levels were measured by flow cytometry. Among metabolic enzymes,we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples,while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity,lowers levels of glycolytic intermediates,decreases the cellular NAD + pool,boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead,we found that LDHA is essential to maintain a correct NAD + /NADH ratio in AML cells. Continuous intracellular NAD + supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively,our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD + /NADH balance in support of their abnormal glycolytic activity and biosynthetic demands,which cannot be compensated for by other cellular NAD + recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells. The online version contains supplementary material available at 10.1186/s40170-025-00392-4. View Publication

Hematologic adverse events are common dose-limiting toxicities in drug development. Classical animal models for preclinical safety assessment of immunotherapies are often limited due to insufficient cross-reactivity with non-human homologous proteins,immune system differences,and ethical considerations. Therefore,we evaluate a human bone marrow (BM) microphysiological system (MPS) for its ability to predict expected hematopoietic liabilities of immunotherapeutics. The BM-MPS consists of a closed microfluidic circuit containing a ceramic scaffold covered with human mesenchymal stromal cells and populated with human BM-derived CD34+ cells in chemically defined growth factor-enriched media. The model supports on-chip differentiation of erythroid,myeloid and NK cells from CD34+ cells over 31 days. The hematopoietic lineage balance and output is responsive to pro-inflammatory factors and cytokines. Treatment with a transferrin receptor-targeting IgG1 antibody results in inhibition of on-chip erythropoiesis. The immunocompetence of the chip is established by the addition of peripheral blood T cells in a fully autologous setup. Treatment with T cell bispecific antibodies induces T cell activation and target cell killing consistent with expected on-target off-tumor toxicities. In conclusion,this study provides a proof-of-concept that this BM-MPS is applicable for in vitro hematopoietic safety profiling of immunotherapeutics. Subject terms: Biologics,Haematopoiesis,Lab-on-a-chip,Drug safety View Publication

Treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) requires new therapy options,especially for patients uneligible for intense chemotherapy or with relapsed or refractory disease. CLEVER-1 is a myeloid checkpoint protein,which can be targeted with a therapeutic function blocking antibody,bexmarilimab. Bexmarilimab has shown clinical efficacy in different solid tumors. Here,we show preclinical data demonstrating expression of CLEVER-1 on immature malignant myeloid cells and their derivates in MDS and AML bone marrow samples and AML cell lines. Highest CLEVER-1 levels were observed in AML with monocytic differentiation. Ex vivo treatment of AML/MDS bone marrow samples with bexmarilimab led to an increase in antigen-presenting human leukocyte antigen DR isotype (HLA-DR) molecule expression. Combination of bexmarilimab with current standard-of-care (SoC) drugs,azacitidine and venetoclax,showed potential for HLA-DR induction and enhanced killing of leukemic cells,respectively. Our non-clinical findings support the feasibility of CLEVER-1 inhibition in AML/MDS to induce antigen presentating molecule expression and potentially,an anti-leukemic effect together with SoC. Therapeutic targeting of CLEVER-1 with bexmarilimab is currently undergoing clinical investigation in the BEXMAB trial ( NCT05428969 ). The online version contains supplementary material available at 10.1038/s41598-025-01675-y. View Publication

Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously,we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice,respectively. In this study,we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system,targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion,and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally,MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture,MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction,pro-oxidant profile amelioration,mitochondrial mass recovery,and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction. View Publication