技术资料

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

In dynamic systems like the circulatory system,establishing localized cytokine gradients is challenging. Upon lipopolysaccharide (LPS) stimulation,we observed that monocytes release numerous migrasomes enriched with inflammatory cytokines,such as TNF-α and IL-6. These cytokines are transported into migrasomes via secretory carriers,leading to their immediate exocytosis or eventual release from detached migrasomes. We successfully isolated TNF-α and IL-6-enriched,monocyte-derived migrasomes from the blood of LPS-treated mice. Total secretion analysis revealed a substantial amount of TNF-α and IL-6 released in a migrasome-packaged form. Thus,detached,monocyte-derived migrasomes represent a type of extracellular vesicle highly enriched with cytokines. Physiologically,these cytokine-laden migrasomes rapidly accumulate at local sites of inflammation,effectively creating a concentrated source of cytokines. Our research uncovers novel mechanisms for cytokine release and delivery,providing new insights into immune response modulation. View Publication

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition associated with the inflammatory activation of alveolar macrophages. Here,we examined the role of circVAPA in regulating inflammasome activation and macrophage inflammatory polarization in an ARDS model.MethodscircVAPA expression levels were analyzed in macrophages isolated from healthy controls and patients with ARDS. In vitro cell models of mouse alveolar macrophages and an in vivo mouse ARDS model were established through Lipopolysaccharide (LPS) stimulation. The effects of circVAPA knockdown on macrophage inflammatory polarization,inflammasome activation,and pulmonary tissue damage were investigated in both cell and animal models. The interaction between circVAPA and downstream factors was verified through a luciferase reporter assay and by silencing circVAPA.ResultscircVAPA upregulation in alveolar macrophages was associated with the inflammation in ARDS patients. circVAPA was also upregulated in LPS-stimulated mouse alveolar macrophages (MH-S cells). Additionally,circVAPA knockdown attenuated the inflammatory activation of MH-S cells and reduced the expression of pyroptosis-related proteins. circVAPA silencing also mitigated the inflammatory effects of LPS-stimulated MH-S cells on lung epithelial cells (MLE-12),and alleviated the inflammatory damage in the pulmonary tissue of ARDS mouse model. We further showed that miR-212-3p/Sirt1 axis mediated the functional role of circVAPA in the inflammatory polarization of MH-S cells.ConclusionOur data suggest that circVAPA promotes inflammasome activity and macrophage inflammation by modulating miR-212-3p/Sirt1 axis in ARDS. Targeting circVAPA may be employed to suppress the inflammatory activation of alveolar macrophages in ARDS.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10735-024-10312-3. 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

IntroductionPlasmacytoid dendritic cells (pDCs) are capable of triggering broad immune responses,yet,their scarcity in blood coupled to their reduced functionality in cancer,makes their therapeutic use for in situ activation or vaccination challenging. MethodsWe designed an in vitro differentiation protocol tailored for human pDCs from cord blood (CB) hematopoietic stem cells (HSCs) with StemRegenin 1 (SR-1) and GM-CSF supplementation. Next,we evaluated the identity and function of CB-pDCs compared to human primary pDCs. Furthermore,we tested the potential of CB-pDCs to support anti-tumor immune responses in co-culture with tumor explants from CRC patients. ResultsHere,we report an in vitro differentiation protocol enabling the generation of 200 pDCs per HSC and highlight the role of GM-CSF and SR-1 in CB-pDC differentiation and function. CB-pDCs exhibited a robust resemblance to primary pDCs phenotypically and functionally. Transcriptomic analysis confirmed strong homology at both,baseline and upon TLR9 or TLR7 stimulation. Further,we could confirm the potential of CB-pDCs to promote inflammation in the tumor microenvironment by eliciting cytokines associated with NK and T cell recruitment and function upon TLR7 stimulation ex vivo in patient tumor explants. DiscussionThis study highlights CB-pDCs as surrogates for primary pDCs to investigate their biology and for their potential use as cell therapy in cancer. 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

Crystalline silica (CS) particle exposure leads to silicosis which is characterized as progressive fibrosis. Fibroblasts are vital effector cells in fibrogenesis. Emerging studies have identified immune sentinel roles for fibroblasts in chronic disease,while their immune-modulatory roles in silicosis remain unclear. Herein,we show that group 2 innate lymphoid cell (ILC2) conversion to ILC1s is closely involved in silicosis progression,which is mediated by activated fibroblasts via interleukin (IL)−18. Mechanistically,Notch3 signaling in mechanics-activated fibroblasts modulates IL-18 production via caspase 1 activity. The mouse-specific Notch3 knockout in fibroblasts retards pulmonary fibrosis progression that is linked to attenuated ILC conversion. Our results indicate that activated fibroblasts in silicotic lungs are regulators of ILC2–ILC1 conversion,associated with silicosis progression via the Notch3–IL-18 signaling axis. This finding broadens our understanding of immune-modulatory mechanisms in silicosis,and indicates potential therapeutic targets for lung fibrotic diseases. Crystalline silica particle exposure in the airways can lead to lung silicosis and progressive fibrosis. Here the authors use mouse silicosis models to show mechanics activated fibroblasts promote conversion of ILC2 to ILC1-like cells pulmonary fibrosis and that this is associated with a Notch3-IL-18 signalling pathway. View Publication

Despite recent advances in immunotherapies targeting single tumor-associated antigens,patients with multiple myeloma eventually relapse. ISB 2001 is a CD3+ T cell engager (TCE) co-targeting BCMA and CD38 designed to improve cytotoxicity against multiple myeloma. Targeting of two tumor-associated antigens by a single TCE resulted in superior cytotoxic potency across a variable range of BCMA and CD38 tumor expression profiles mimicking natural tumor heterogeneity,improved resistance to competing soluble factors and exhibited superior cytotoxic potency on patient-derived samples and in mouse models. Despite the broad expression of CD38 across human tissues,ISB 2001 demonstrated a reduced T cell activation profile in the absence of tumor cells when compared to TCEs targeting CD38 only. To determine an optimal first-in-human dose for the ongoing clinical trial (NCT05862012),we developed an innovative quantitative systems pharmacology model leveraging preclinical data,using a minimum pharmacologically active dose approach,therefore reducing patient exposure to subefficacious doses of therapies. Perro and colleagues develop a CD3+ T cell engager co-targeting BCMA and CD38 to improve immunotherapy for multiple myeloma,demonstrate cytotoxicity in patient-derived samples and murine models and develop a quantitative systems pharmacology model. View Publication

Centrioles define centrosome structure and function. Deregulation of centriole numbers can cause developmental defects and cancer. The p53 tumor suppressor limits the growth of cells lacking or harboring additional centrosomes and can be engaged by the “mitotic surveillance” or the “PIDDosome pathway”,respectively. Here,we show that early B cell progenitors frequently present extra centrioles,ensuing their high proliferative activity and related DNA damage. Extra centrioles are efficiently cleared during B cell maturation. In contrast,centriole loss upon Polo-like kinase 4 (Plk4) deletion causes apoptosis and arrests B cell development. This defect can be rescued by co-deletion of Usp28,a critical component of the mitotic surveillance pathway,that restores cell survival and maturation. Centriole-deficient mature B cells are proliferation competent and mount a humoral immune response. Our findings imply that progenitor B cells are intolerant to centriole loss but permissive to centriole amplification,a feature potentially facilitating their malignant transformation. Centrioles organize chromosome segregation,migration,and the immune synapse. Here,Schapfl et al. show that B cell progenitors tolerate centriole overamplification,but not loss,while mature B cells can mount a humoral immune response in their absence. View Publication