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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

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety,however,is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1,but none has quantitatively defined the safety level of transplant therapies. Here,using genome-engineering strategies,we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore,we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here,we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic. View Publication

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca-1) cells to reconstitute aged BM and rejuvenate the aged heart,and examined the underlying molecular mechanisms. BM Sca-1+ or Sca-1- cells from young (2-3 months) or aged (18-19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca-1+,young Sca-1-,old Sca-1+,and old Sca-1- . Four months later,expression of rejuvenation-related genes (Bmi1,Cbx8,PNUTS,Sirt1,Sirt2,Sirt6) and proteins (CDK2,CDK4) was increased along with telomerase activity and telomerase-related protein (DNA-PKcs,TRF-2) expression,whereas expression of senescence-related genes (p16INK4a,P19ARF,p27Kip1 ) and proteins (p16INK4a,p27Kip1 ) was decreased in Sca-1+ chimeric hearts,especially in the young group. Host cardiac endothelial cells (GFP- CD31+ ) but not cardiomyocytes were the primary cell type rejuvenated by young Sca-1+ cells as shown by improved proliferation,migration,and tubular formation abilities. C-X-C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP+ ) cells isolated from young Sca-1+ chimeric hearts. Protein expression of Cxcr4,phospho-Akt,and phospho-FoxO3a in endothelial cells derived from the aged chimeric heart was increased,especially in the young Sca-1+ group. Reconstitution of aged BM with young Sca-1+ cells resulted in effective homing of functional stem cells in the aged heart. These young,regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells. View Publication

Intestinal fibrosis is a serious complication in inflammatory bowel disease (IBD). Despite the remarkable success of recent anti-inflammatory therapies for IBD,incidence of intestinal fibrosis and need for bowel resection have not significantly changed. To clarify the contribution of haematopoietic-derived cells in intestinal fibrosis,we prepared bone marrow (BM) chimeric mice (chimeras),which were reconstituted with BM cells derived from enhanced green fluorescent protein (EGFP)-transgenic mice or CC chemokine receptor 2 (CCR2)-deficient mice. After 2 months of transplantation,BM chimeras were treated with azoxymethane/dextran sodium sulphate. During chronic inflammation,CCR2+ BM-derived monocyte and fibrocyte infiltration into the colon and CC chemokine ligand 2 production increased,leading to colon fibrosis in EGFP BM chimeras. In CCR2-deficient BM chimeras,monocyte and fibrocyte numbers in the colonic lamina propria significantly decreased,and colon fibrosis was attenuated. In colon tissue,mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 but not of collagen I,transforming growth factor-beta1 or matrix metalloproteinases was significantly different between the two chimeras. CCR2+ monocytes and fibrocytes showed high Timp1 mRNA expression. Our results suggest that infiltrating CCR2+ monocytes and their progenies,fibrocytes,promote colon fibrosis by inhibiting collagen degradation through TIMP-1 production. View Publication

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens,but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells,we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs,40{\%} of antigen-specific SED B cells bind antigen within 2 h,whereas unspecifc cells do not,indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice,but is unperturbed in mice depleted of classical dendritic cells (DC). Thus,we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses,and should be taken into account when developing mucosal vaccines. View Publication

Deregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is found in cancer with STAT5A/B controlling leukemic cell survival and disease progression. As mutations in STAT5B,but not STAT5A,have been frequently described in hematopoietic tumors,we used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The absence of STAT5A decreased cell survival and colony formation. Even more drastic effects were observed in the absence of STAT5B. STAT5B-deficient cells formed BCR/ABL+ colonies or stable cell lines at low frequency. The rarely evolving Stat5b-/- cell lines expressed enhanced levels of BCR/ABL oncoprotein compared to wild-type cells. In line,Stat5b-/- leukemic cells induced leukemia with a significantly prolonged disease onset,whereas Stat5a-/- cells rapidly caused a fatal disease superimposable to wild-type cells. RNA-sequencing (RNA-seq) profiling revealed a marked enhancement of interferon (IFN)-alpha and IFN-gamma signatures in Stat5b-/- cells. Inhibition of IFN responses rescued BCR/ABL+ colony formation of Stat5b-/--deficient cells. A downregulated IFN response was also observed in patients suffering from leukemia carrying STAT5B mutations. Our data define STAT5B as major STAT5 isoform driving BCR/ABL+ leukemia. STAT5B enables transformation by suppressing IFN-alpha/gamma,thereby facilitating leukemogenesis. Our findings might help explain the high frequency of STAT5B mutations in hematopoietic tumors. View Publication

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation,we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range,enabling targeting of many previously inaccessible PAMs. On average,enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a,and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing,endogenous gene activation and C-to-T base editing,and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively,enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing. View Publication