技术资料

Natural killer (NK) cells are innate immune cells that are crucial for anticancer activity and have been developed as an immune cell therapy for leukemia. However,their limited effectiveness against solid tumors has prompted research into methods to enhance NK cell activity through combination therapies. Health supplements capable of boosting immune surveillance against tumor cells are gaining attention owing to their potential benefits. Resveratrol,a stilbenoid produced by several plants including peanuts and grapes,reportedly exerts anticancer effects and can activate immune cells. The peanut sprout extract cultivated with fermented sawdust medium (PSEFS) is rich in resveratrol,leveraging its health benefits in terms of the dry weight of herbal products,thus maximizing the utilization of resveratrol’s beneficial properties. Our study compared the efficacy of resveratrol and PSEFS and revealed that PSEFS significantly enhanced NK cell activation compared with an equivalent dose of resveratrol. We investigated the ability of PSEFS to potentiate NK cell anticancer activity,focusing on NK cell survival,tumor cell lysis,and NK cell activation in PSEFS-administered mice. Our findings suggest that PSEFS could be a potential NK cell booster for cancer immunotherapy. View Publication

Immune dysregulation-induced inflammation serves as a driving force in the progression of metabolic dysfunction-associated steatohepatitis (MASH),while the underlying cellular and molecular mechanisms remain largely uncharted. A Western diet (WD) is employed to construct mouse models of metabolic dysfunction associated steatotic liver disease (MASLD) or MASH. Mass cytometry identifies a c-kit+ cDC1 subset whose frequency is reduced in the livers of mice and patients with MASH compared with healthy controls. Adoptive cell transfer of c-kit+ cDC1 protects the progression of MASH. Moreover,analysis of gut microbe sequence shows that WD-fed mice and MASLD/MASH patients exhibit gut microbiota dysbiosis,with an elevated abundance of H2S-producing Desulfovibrio_sp. Transplanting of MASH-derived fecal flora,Desulfovibrio_sp.,or injecting H2S intraperitoneally into MASLD mice decreases the c-kit+cDC1 population and exacerbates liver inflammation. Mechanistically,H2S induces autophagic cell death of cDC1 in a c-kit-dependent manner in cDC-specific c-kit-/- and Atg5-/- mice. We thus uncover that microbiota-derived H2S triggers the autophagic cell death of c-kit+ cDC1 and ignites the liver inflammatory cascade in MASH. The immune regulatory mechanism for metabolic dysfunction-associated steatohepatitis (MASH) remains elusive. Here,the authors identify a c-kit+ cDC1 subset,which can be depleted by Desulfovibrio_sp.-induced H2S via autophagic cell death and contributing to uncontrolled inflammation for MASH progression. View Publication

Characterizing the HIV-1 reservoir in blood and tissues is crucial for the development of curative strategies. Using an HIV Tat mRNA-containing lipid nanoparticle (Tat-LNP) in combination with panobinostat,we show that p24+ cells from blood and lymph nodes exhibit distinct phenotypes. Blood p24+ cells are found in both central/transitional (TCM/TTM) and effector memory subsets,mostly lack CXCR5 expression and are enriched in GZMA+ cells. In contrast,most lymph node p24+ cells display a TCM/TTM phenotype,with approximately 50% expressing CXCR5 and nearly all lacking GZMA expression. Furthermore,germinal center T follicular helper cells do not appear to harbor the translation-competent reservoir in long-term suppressed individuals. Near full-length HIV-1 sequencing in longitudinal samples from matched blood,lymph nodes,and gut indicates that clones of infected cells,including those carrying an inducible provirus,persist and spread across various anatomical compartments. Finally,uniform genetic diversity across sites suggests the absence of ongoing replication in tissues under treatment. Here,Pardons and Lambrechts et al show that HIV-1 reservoirs in blood and lymph nodes differ phenotypically. Furthermore, germinal center T follicular helper cells do not harbor the inducible reservoir in long-term suppressed individuals. Infected clones can spread across tissues and persist without active replication. View Publication

AbstractSingle-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here,we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length,as they mostly originate from the subtelomere,but can be used as a biomarker for chromatin condensation. Using long-read sequencing,we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence,contrary to common belief. We provide a new tool,Telomemore,which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases,we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred,and how it complements common RNA-seq-based cell cycle inference,which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox. Graphical Abstract Graphical Abstract View Publication

AbstractBackgroundThe complex aetiology of type 1 diabetes (T1D),characterised by a detrimental cross‐talk between the immune system and insulin‐producing beta cells,has hindered the development of effective disease‐modifying therapies. The discovery that the pharmacological activation of LRH‐1/NR5A2 can reverse hyperglycaemia in mouse models of T1D by attenuating the autoimmune attack coupled to beta cell survival/regeneration prompted us to investigate whether immune tolerisation could be translated to individuals with T1D by LRH‐1/NR5A2 activation and improve islet survival.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from individuals with and without T1D and derived into various immune cells,including macrophages and dendritic cells. Cell subpopulations were then treated or not with BL001,a pharmacological agonist of LRH‐1/NR5A2,and processed for: (1) Cell surface marker profiling,(2) cytokine secretome profiling,(3) autologous T‐cell proliferation,(4) RNAseq and (5) proteomic analysis. BL001‐target gene expression levels were confirmed by quantitative PCR. Mitochondrial function was evaluated through the measurement of oxygen consumption rate using a Seahorse XF analyser. Co‐cultures of PBMCs and iPSCs‐derived islet organoids were performed to assess the impact of BL001 on beta cell viability.ResultsLRH‐1/NR5A2 activation induced a genetic and immunometabolic reprogramming of T1D immune cells,marked by reduced pro‐inflammatory markers and cytokine secretion,along with enhanced mitohormesis in pro‐inflammatory M1 macrophages and mitochondrial turnover in mature dendritic cells. These changes induced a shift from a pro‐inflammatory to an anti‐inflammatory/tolerogenic state,resulting in the inhibition of CD4+ and CD8+ T‐cell proliferation. BL001 treatment also increased CD4+/CD25+/FoxP3+ regulatory T‐cells and Th2 cells within PBMCs while decreasing CD8+ T‐cell proliferation. Additionally,BL001 alleviated PBMC‐induced apoptosis and maintained insulin expression in human iPSC‐derived islet organoids.ConclusionThese findings demonstrate the potential of LRH‐1/NR5A2 activation to modulate immune responses and support beta cell viability in T1D,suggesting a new therapeutic approach.Key Points LRH‐1/NR5A2 activation in inflammatory cells of individuals with type 1 diabetes (T1D) reduces pro‐inflammatory cell surface markers and cytokine release.LRH‐1/NR5A2 promotes a mitohormesis‐induced immuno‐resistant phenotype to pro‐inflammatory macrophages.Mature dendritic cells acquire a tolerogenic phenotype via LRH‐1/NR5A2‐stimulated mitochondria turnover.LRH‐1/NR5A2 agonistic activation expands a CD4+/CD25+/FoxP3+ T‐cell subpopulation.Pharmacological activation of LRH‐1/NR5A2 improves the survival iPSC‐islets‐like organoids co‐cultured with PBMCs from individuals with T1D. LRH‐1/NR5A2 activation in inflammatory cells of individuals with type 1 diabetes (T1D) reduces pro‐inflammatory cell surface markers and cytokine release.LRH‐1/NR5A2 promotes a mitohormesis‐induced immuno‐resistant phenotype to pro‐inflammatory macrophages.Mature dendritic cells acquire a tolerogenic phenotype via LRH‐1/NR5A2‐stimulated mitochondria turnover.LRH‐1/NR5A2 agonistic activation expands a CD4+/CD25+/FoxP3+ T‐cell subpopulation.Pharmacological activation of LRH‐1/NR5A2 improves the survival iPSC‐islets‐like organoids co‐cultured with PBMCs from individuals with T1D. View Publication

The role of the bone marrow (BM) microenvironment in regulating the antitumor immune response in Waldenstrom macroglobulinemia (WM) remains poorly understood. Here we transcriptionally and phenotypically profiled non-malignant (CD19- CD138-) BM cells from WM patients with a focus on myeloid derived suppressive cells (MDSCs) to provide a deeper understanding of their role in WM. We found that HLA-DRlowCD11b+CD33+ MDSCs were significantly increased in WM patients as compared to normal controls,with an expansion of predominantly polymorphonuclear (PMN)-MDSCs. Single-cell immunogenomic profiling of WM MDSCs identified an immune-suppressive gene signature with upregulated inflammatory pathways associated with interferon and tumor necrosis factor (TNF) signaling. Gene signatures associated with an inflammatory and immune suppressive environment were predominately expressed in PMN-MDSCs. In vitro,WM PMN-MDSCs demonstrated robust T-cell suppression and their viability and expansion was notably enhanced by granulocyte colony stimulating factor (G-CSF) and TNFα. Furthermore,BM malignant B-cells attracted PMN-MDSCs to a greater degree than monocytic MDSCs. Collectively,these data suggest that malignant WM B cells actively recruit PMN-MDSCs which promote an immunosuppressive BM microenvironment through a direct T cell inhibition,while release of G-CSF/TNFα in the microenvironment further promotes PMN-MDSC expansion and in turn immune suppression. Targeting PMN-MDSCs may therefore represent a potential therapeutic strategy in patients with WM. View Publication

This study aimed to identify unique characteristics in the peripheral blood mononuclear cells (PBMCs) of endometriosis patients and develop a non-invasive early diagnostic tool. Using single-cell RNA sequencing (scRNA-seq),we constructed the first single-cell atlas of PBMCs from endometriosis patients based on 107,964 cells and 25,847 genes. Within CD16+ monocytes,we discovered JUP as a dysregulated gene. To assess its diagnostic potential,we measured peritoneal fluid (PF) and serum JUP levels in a large cohort of 199 patients including 20 women with ovarian cancer (OC). JUP was barely detectable in PF but was significantly elevated in the serum of patients with endometriosis and OC,with levels 1.33 and 2.34 times higher than controls,respectively. Additionally,JUP was found in conditioned culture media of CD14+/CD16+ monocytes aligning with our scRNA-seq data. Serum JUP levels correlated with endometriosis severity and endometrioma presence but were unaffected by dysmenorrhea,menstrual cycle,or adenomyosis. When combined with CA125 (cancer antigen 125) JUP enhanced the specificity of endometriosis diagnosis from 89.13% (CA125 measured alone) to 100%. While sensitivity remains a challenge at 19%,our results suggest that JUP’s potential to enhance diagnostic accuracy warrants additional investigation. Furthermore,employing serum JUP as a stratification marker unlocked the potential to identify additional endometriosis-related genes,offering novel insights into disease pathogenesis. View Publication

Despite significant advancements in targeted- and immunotherapies,millions of patients with cancer still succumb to the disease each year. In renal cell carcinoma,up to 25% of metastatic patients do not respond to first-line therapies. This reality underscores the urgent need for innovative or repurposed therapies to effectively treat these patients. Patient-derived organoids represent a promising model for evaluating treatment efficacy and toxicity,offering a potential breakthrough in personalized medicine. However,utilizing organoid models for drug screening presents several challenges. Our protocol aims to address these obstacles by outlining a practical approach to successfully isolate and cultivate patient-derived renal cell carcinoma and kidney organoids for treatment screening purposes. Graphical abstract Patient-derived organoids represent a promising model for evaluating treatment efficacy and toxicity,offering a potential breakthrough in personalized medicine. Nowak-Sliwinska and colleagues present a detailed protocol for obtaining kidney and kidney cancer organoids for drug development and precision oncology. View Publication

Germinal center (GC) formation,which is an integrant part of humoral immunity,involves energy-consuming metabolic reprogramming. Rag-GTPases are known to signal amino acid availability to cellular pathways that regulate nutrient distribution such as the mechanistic target of rapamycin complex 1 (mTORC1) pathway and the transcription factors TFEB and TFE3. However,the contribution of these factors to humoral immunity remains undefined. Here,we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs,produce antibodies,and to generate plasmablasts during both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically,the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells,which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development,GC formation in Peyer’s patches and TI humoral immunity,but not TD humoral immunity in the absence of Rag-GTPases. Collectively,our data establish the Rag GTPase-TFEB/TFE3 pathway as a likely mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells. Rag-GTPases play roles in sensing nutrient availability,and it is not fully known how they contribute to energy-consuming immunological processes such as the B cell response. Here authors show that genomic deletion fo RagA/RagB distrupts both T-dependent and T-independent humoral immune responses,independent of mechanistic target of rapamycin complex 1 but involving the transcription factors TFEB and TFE3. View Publication

Ginseng may improve the myelosuppression and intestinal microbiota disorder induced by cyclophosphamide (CY); however,the effect of ginseng components on hematopoietic stem cell (HSC) damage remains largely unexplored. The present study aimed to assess the protective effect of ginseng extract (GE),total ginsenosides (TG) and total polysaccharides (TP) from ginseng on the intestinal microflora and HSCs of model mice. In the present study,a mouse model of HSC damage induced by CY was constructed,intestinal microflora of fecal samples were sequenced using the 16S ribosomal RNA (rRNA) sequencing techniques,the differentially expressed genes (DEGs) of HSCs were analyzed using high-throughput RNA-sequencing,cell apoptosis and erythroid differentiation were detected using flow cytometry and the blood cell parameters were analyzed using a hematology analyzer. Analysis of the 16S rRNA in fecal samples showed that GE,TG and TP improved an imbalanced intestinal microflora,where the relative abundance of Lactobacillus intestinalis had a positive correlation with ginsenosides content. Specifically,TP significantly increased the expression of low-abundance microflora. Transcriptomic analysis results revealed 2,250,3,432 and 261 DEGs in the GE,TG and TP groups compared with those in the Model group,respectively. In the expression analysis of DEGs,both TG and GE were found to markedly increase the expression levels of Klf4,Hhex,Pbx1,Kmt2a,Mecom,Zc3h12a,Zbtb16,Lilr4b,Flt3 and Klf13. Furthermore,TG inhibited the apoptosis of HSCs by increasing the expression levels of Bcl2 and Mcl1,whilst decreasing the expression of Bax. By contrast,GE inhibited the apoptosis of HSCs by reducing the expression of Bax and Bad. Regarding erythroid differentiation and blood cell parameters,GE was found to significantly increase the expression of TER-119. In addition,GE and TG improved all blood cell parameters,including the count of white blood cells,neutrophils (NEUT),lymphocytes (LYMPH),red blood cells (RBC),hemoglobin (HGB) and reticulocyte and platelets (PLT),whereas TP could only improve the counts of LYMPH,RBC,HGB and PLT. The improvement effect of GE and TG on WBC,NEUT and Ret was superior to TP. In conclusion,TG may protect the hematopoiesis function of HSCs in a CY-induced mouse model of HSC damage,followed by GE. However,TP did not appear to improve HSC damage. Ginsenosides may therefore be considered essential ingredients in GE when protecting HSCs against damage. GE and TG exerted their protective effects on HSCs by inhibiting the apoptosis of HSCs whilst improving the imbalance of intestinal microflora. View Publication

Primary antiphospholipid syndrome (PAPS) is a life-threatening clotting disorder mediated by pathogenic autoantibodies. Here we dissect the origin of self-reactive B cells in human PAPS using peripheral blood and bone marrow of patients with triple-positive PAPS via combined single-cell RNA sequencing,B cell receptors (BCR) repertoire profiling,CITEseq analysis and single cell immortalization. We find that antiphospholipid (aPL)-specific B cells are present in the naive compartment,polyreactive,and derived from the natural repertoire. Furthermore,B cells with aPL specificities are not eliminated in patients with PAPS,persist until the memory and long-lived plasma cell stages,likely after defective germinal center selection,while becoming less polyreactive. Lastly,compared with the non-PAPS cells,PAPS B cells exhibit distinct IFN and APRIL signature as well as dysregulated mTORC1 and MYC pathways. Our findings may thus elucidate the survival mechanisms of these autoreactive B cells and suggest potential therapeutic targets for the treatment of PAPS. Primary antiphospholipid syndrome (PAPS) is a clotting disorder attributed to autoreactive antibodies produced by B cells. Here the authors show,using single cell omics and B cell repertoire data,that autoreactive B cells originate from the natural B cell repertoire and escape germinal center selection to persist in PAPS patient via potential dysregulation of mTORC1 and MYC pathways. View Publication

SummaryMonocytes are critical to innate immunity,participating in chemotaxis during tissue injury,infection,and inflammatory conditions. However,the migration dynamics of human monocytes under different guidance cues are not well characterized. Here,we developed a microfluidic device to profile the migration characteristics of human monocytes under chemotactic and barotactic guidance cues while also assessing the effects of age and cytokine stimulation. Human monocytes preferentially migrated toward the CCL2 gradient through confined microchannels,regardless of donor age and migration pathway. Stimulation with interferon (IFN)-γ,but not granulocyte-macrophage colony-stimulating factor (GM-CSF),disrupted monocyte navigation through complex paths and decreased monocyte CCL2 chemotaxis,velocity,and CCR2 expression. Additionally,monocytes exhibited a bias toward low-hydraulic-resistance pathways in asymmetric environments,which remained consistent across donor ages,cytokine stimulation,and chemoattractants. This microfluidic system provides insights into the unique migratory behaviors of human monocytes and is a valuable tool for studying peripheral immune cell migration in health and disease. Graphical abstract Highlights•The MAP chip profiles migration of human monocytes under various chemotactic and barotactic cues•Monocytes preferentially migrate toward CCL2 gradients,regardless of migration pathway and donor age•IFN-γ reduces human monocyte chemotaxis,velocity,and CCR2 expression•Human monocytes show biased migration toward low-hydraulic-resistance pathways MotivationCell migration is fundamental to the biological processes that drive health and disease. While in vivo models provide invaluable insights into cell migration within complex biological environments,precise control over the microenvironment and single-cell tracking is essential to deepen our understanding of the fundamental characteristics of cell migration. We present a high-throughput microfluidic platform,termed the migration analysis of peripheral immune cells (MAP) chip,that features four distinct sets of microchannels designed to assess the effects of both chemotactic and barotactic stimuli on cell migration at a single-cell level. By profiling human monocyte migration using the MAP chip,we demonstrated the utility of this device in characterizing migration of human monocytes under diverse conditions. Hall et al. introduce the MAP chip,a microfluidic platform for profiling human monocytes under chemotactic and barotactic guidance cues. It reveals biased migration toward low-hydraulic-resistance pathways,disrupted migration upon cytokine stimulation,and consistent chemotaxis and barotaxis across donor ages—enhancing our understanding of human monocyte migration characteristics. View Publication