技术资料

A large body of evidence suggests that B-cell lymphomas with enhanced Myc expression are associated with an aggressive phenotype and poor prognosis,which makes Myc a compelling therapeutic target. Phosphodiesterase 4B (PDE4B),a main hydrolyzer of cyclic AMP (cAMP) in B cells,was shown to be involved in cell survival and drug resistance in diffuse large B cell lymphomas (DLBCL). However,the interrelationship between Myc and PDE4B remains unclear. Here,we first demonstrate the presence of the Myc-PDE4B feed-forward loop,in which Myc and PDE4B mutually reinforce the expression of each other. Next,the combined targeting of Myc and PDE4 synergistically prevented the proliferation and survival of B lymphoma cells in vitro and in a mouse xenograft model. We finally recapitulated this combinatorial effect in Emu-myc transgenic mice; co-inhibition of Myc and PDE4 suppressed lymphomagenesis and restored B cell development to the wild type level that was associated with marked reduction in Myc levels,unveiling the critical role of the Myc-PDE4B amplification loop in the regulation of Myc expression and the pathogenesis of B cell lymphoma. These findings suggest that the disruption of the Myc-PDE4B circuitry can be exploited in the treatment of B cell malignancies. View Publication

Serrated adenocarcinoma,an alternative pathway for colorectal cancer (CRC) development,accounts for 15{\%}-30{\%} of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C zeta (PKCzeta) and PKClambda/iota was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKClambda/iota deficiency led to immunogenic cell death and the infiltration of CD8+ T cells,which repressed tumor initiation,PKCzeta loss impaired interferon and CD8+ T cell responses,which resulted in tumorigenesis. Combined treatment with a TGF-beta receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC. View Publication

This study aimed to determine the association between non-high-fat diet-induced obesity- (non-DIO-) associated gut microbiome dysbiosis with gut abnormalities like cellular turnover of intestinal cells,tight junctions,and mucin formation that can impact gut permeability. We used leptin-deficient (Lepob/ob) mice in comparison to C57BL/6J control mice,which are fed on identical diets,and performed comparative and correlative analyses of gut microbiome composition,gut permeability,intestinal structural changes,tight junction-mucin formation,cellular turnover,and stemness genes. We found that obesity impacted cellular turnover of the intestine with increased cell death and cell survival/proliferation gene expression with enhanced stemness,which are associated with increased intestinal permeability,changes in villi/crypt length,and decreased expression of tight junctions and mucus synthesis genes along with dysbiotic gut microbiome signature. Obesity-induced gut microbiome dysbiosis is also associated with abnormal intestinal organoid formation characterized with decreased budding and higher stemness. Results suggest that non-DIO-associated gut microbiome dysbiosis is associated with changes in the intestinal cell death versus cell proliferation homeostasis and functions to control tight junctions and mucous synthesis-regulating gut permeability. View Publication

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and posses considerable immunomodulatory potential. MSCs have been shown to promote allograft survival,yet the mechanisms behind this phenomenon have not been fully defined. Here,we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation,we report that MSCs promote graft survival in an HGF-dependent manner. Moreover,our data indicate that topically administered recombinant HGF (1) suppresses antigen-presenting cell maturation in draining lymphoid tissue,(2) limits T-helper type-1 cell generation,(3) decreases inflammatory cell infiltration into grafted tissue,and (4) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019. View Publication

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities,but the detailed,underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU),which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells,respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice,the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis,but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50{\%} of enterochromaffin (EC) cells expressed 5-HT3AR,but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast,plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion,5-HT3R signaling may enhance 5-HT release,possibly from EC cells intravascularly,or paracrine,resulting in increases in plasma 5-HT concentration,which in turn,enhances apoptotic activities induced by 5-FU.-Mikawa,S.,Kondo,M.,Kaji,N.,Mihara,T.,Yoshitake,R.,Nakagawa,T.,Takamoto,M.,Nishimura,R.,Shimada,S.,Ozaki,H.,Hori,M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-alpha production by enhancing serotonin release from enterochromaffin cells. View Publication

The complex process of platelet formation originates with the hematopoietic stem cell,which differentiates through the myeloid lineage,matures,and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRalpha+ perivascular cells,derived from human umbilical cord tissue,which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression,yielding platelets that are functional with low basal activation levels,a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo. View Publication

Soybeans are a rich source of isoflavones that have been linked with anti-inflammatory processes and various health benefits. However,specific mechanisms whereby soy bioactives impact immune cell subsets are unclear. Isoflavones,such as genistein and daidzein,are metabolized by microbes to bioactive metabolites as O-desmethylangolensin (O-DMA) and equol,whose presence has been linked to health benefits. We examined how soy isoflavones and metabolites impact natural killer (NK) cell signaling and function. We observe no impact of isoflavones on viability of healthy donor peripheral blood mononuclear cells (PBMCs) or NK cells,even at high (25 µM) concentrations. However,pre-treatment of PBMCs with physiologically-relevant concentrations of genistein (p = 0.0023) and equol (p = 0.006) decreases interleukin (IL)-12/IL-18-induced interferon-gamma (IFN-gamma) production versus controls. Detailed cellular analyses indicate genistein and equol decrease IL-12/IL-18-induced IFN-gamma production by human NK cell subsets,but do not consistently alter cytotoxicity. At the level of signal transduction,genistein decreases IL-12/IL-18-induced total phosphorylated tyrosine,and phosphorylation MAPK pathway components. Further,genistein limits IL-12/IL-18-mediated upregulation of IL-18Ralpha expression on NK cells (p = 0.0109). Finally,in vivo studies revealed that C57BL/6 mice fed a soy-enriched diet produce less plasma IFN-gamma following administration of IL-12/IL-18 versus control-fed animals (p {\textless} 0.0001). This study provides insight into how dietary soy modulates NK cell functions. View Publication

Circulating Tumor Cells (CTCs) represent an easy,repeatable and representative access to information regarding solid tumors. However,their detection remains difficult because of their paucity,their short half-life,and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast,sensitive and affordable technique,ideal for rare cells detection. Adapted to CTCs detection (i.e. extremely rare cells),most FC-based techniques require a time-consuming pre-enrichment step,followed by a 2-hours staining procedure,impeding on the efficiency of CTCs detection. We overcame these caveats and reduced the procedure to less than one hour,with minimal manipulation. First,cells were simultaneously fixed,permeabilized,then stained. Second,using low-speed FC acquisition conditions and two discriminators (cell size and pan-cytokeratin expression),we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases,this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses. View Publication

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs),the brain cells which differentiate to myelin-forming cells,microglia participate in both myelin injury and remyelination during multiple sclerosis. However,the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here,we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination,whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs,cultured either alone or with astrocytes,to inflammatory EVs,we observed a blockade of OPC maturation only in the presence of astrocytes,implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo,as indicated by immunohistochemical and qPCR analyses,whereas surface lipid components of EVs promote OPC migration and/or differentiation,linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined,we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration,the first fundamental step in myelin repair. From this study,microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions,which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis,but also in neurological and neuropsychiatric diseases characterized by demyelination. View Publication

Clonal hematopoiesis (CH) is a common aging-associated condition with increased risk of hematologic malignancy. Knowledge of the mechanisms driving evolution from CH to overt malignancy has been hampered by a lack of in vivo models that orthogonally activate mutant alleles. Here,we develop independently regulatable mutations in DNA methyltransferase 3A (Dnmt3a) and nucleophosmin 1 (Npm1),observed in human CH and AML,respectively. We find Dnmt3a mutation expands hematopoietic stem and multipotent progenitor cells (HSC/MPPs),modeling CH. Induction of mutant Npm1 after development of Dnmt3a-mutant CH causes progression to myeloproliferative disorder (MPD),and more aggressive MPD is observed with longer latency between mutations. MPDs uniformly progress to acute myeloid leukemia (AML) following transplant,accompanied by a decrease in HSC/MPPs and an increase in myeloid-restricted progenitors,the latter of which propagate AML in tertiary recipient mice. At a molecular level,progression of CH to MPD is accompanied by selection for mutations activating Ras/Raf/MAPK signaling. Progression to AML is characterized by additional oncogenic signaling mutations (Ptpn11,Pik3r1,Flt3) and/or mutations in epigenetic regulators (Hdac1,Idh1,Arid1a). Together,our study demonstrates that Npm1 mutation drives evolution of Dnmt3a-mutant CH to AML and rate of disease progression is accelerated with longer latency of CH. View Publication

The flow cytometric crossmatch (FCXM) assay,which detects the presence of donor specific HLA antibodies in patient sera,is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time,we developed and validated two modified crossmatch procedures,namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60{\%} and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform,reduced wash times,increased serum to cell suspension volume ratio,shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification,employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63{\%} vs 69 ± 19.06{\%}),reduce cell isolation time (by ∼40{\%}) and conserve FCXM assay reagents. Importantly,linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98-0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally,a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7{\%} and 96.8{\%} specificity and sensitivity,respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care. View Publication

BACKGROUND The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here,we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS EXID's clinical and immunological characteristics were compared to immunological responders (IRs),immunological nonresponders (INRs),healthy controls (HCs),and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/mul,compared with CD4+ increases of 193 cells/mul and 427 cells/mul in INR and IR,respectively. EXID had reduced naive CD4+ T cells,but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR,but the IL-7 axis was profoundly perturbed compared with HC,IR,INR,and ICL. Genes involved in T cell and monocyte/macrophage function,autophagy,and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC,while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS EXID is a distinct immunological outcome compared with previously described INR. Anti-CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID. View Publication