技术资料

Recurrent oncogenic mutations of MyD88 have been identified in a variety of lymphoid malignancies. Gain-of-function mutations of MyD88 constitutively activate downstream NF-$\kappa$B signaling pathways,resulting in increased cellular proliferation and survival. However,whether MyD88 activity can be aberrantly regulated in MyD88-wild-type lymphoid malignancies remains poorly understood. SPOP is an adaptor protein of CUL3-based E3 ubiquitin ligase complex and frequently mutated genes in prostate and endometrial cancers. In this study,we reveal that SPOP binds to and induces the nondegradative ubiquitination of MyD88 by recognizing an atypical SPOP-binding motif in MyD88. This modification blocks Myddosome assembly and downstream NF-$\kappa$B activation. SPOP is mutated in a subset of lymphoid malignancies,including diffuse large B-cell lymphoma (DLBCL). Lymphoid malignancies-associated SPOP mutants exhibited impaired binding to MyD88 and suppression of NF-$\kappa$B activation. The DLBCL-associated,SPOP-binding defective mutants of MyD88 escaped from SPOP-mediated ubiquitination,and their effect on NF-$\kappa$B activation is stronger than that of wild-type MyD88. Moreover,SPOP suppresses DLBCL cell growth in vitro and tumor xenograft in vivo by inhibiting the MyD88/NF-$\kappa$B signaling. Therefore,SPOP acts as a tumor suppressor in DLBCL. Mutations in the SPOP-MyD88 binding interface may disrupt the SPOP-MyD88 regulatory axis and promote aberrant MyD88/NF-$\kappa$B activation and cell growth in DLCBL. View Publication

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential,it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin,expressed on the surfaces of multiple cell types,possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore,we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-,CD45-,and CD11b/c-negative and CD90-,CD29-,CD44-,CD54-,CD73-,and CD105-positive. Osteoblast,adipocyte,and chondrocyte differentiation was observed in these cells. In total,yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly,MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106,whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover,senescence-associated $\beta$-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs. View Publication

Therapeutic drug monitoring is routinely performed to maintain optimal tacrolimus concentrations in kidney transplant recipients. Nonetheless,toxicity and rejection still occur within an acceptable concentration-range. To have a better understanding of the relationship between tacrolimus dose,tacrolimus concentration,and its effect on the target cell,we developed functional immune tests for the quantification of the tacrolimus effect. Twelve healthy volunteers received a single dose of tacrolimus,after which intracellular and whole blood tacrolimus concentrations were measured and were related to T cell functionality. A significant correlation was found between tacrolimus concentrations in T cells and whole blood concentrations (r = 0.71,p = 0.009),while no correlation was found between tacrolimus concentrations in peripheral blood mononuclear cells (PBMCs) and whole blood (r = 0.35,p = 0.27). Phytohemagglutinin (PHA) induced the production of IL-2 and IFN$\gamma$,as well as the inhibition of CD71 and CD154 expression on T cells at 1.5 h post-dose,when maximum tacrolimus levels were observed. Moreover,the in vitro tacrolimus effect of the mentioned markers corresponded with the ex vivo effect after dosing. In conclusion,our results showed that intracellular tacrolimus concentrations mimic whole blood concentrations,and that PHA-induced cytokine production (IL-2 and IFN$\gamma$) and activation marker expression (CD71 and CD154) are suitable readout measures to measure the immunosuppressive effect of tacrolimus on the T cell. View Publication

We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C,while cystic organoids can survive up to 48 h. We find that in the presence of AFGPs,the organoid survival is prolonged up to 72 h,irrespective of their developmental stage. Fluorescence microscopy experiments reveal that the AFGPs predominately localize at the cell surface and cover the cell membranes. Our findings support a mechanism in which the positive effect of AFGPs on cell survival during hypothermic storage involves the direct interaction of AFGPs with the cell membrane. Our research highlights organoids as an attractive multicellular model system for studying the action of AFGPs that bridges the gap between single-cell and whole-organ studies. View Publication

Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data,we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development,which can be linked to functional maturation of typical end-stage blood neutrophil killing activities. View Publication

Early studies in exercise immunology suggested acute bouts of exercise had an immunosuppressive effect in human subjects. However,recent data,show acute bouts of combined aerobic and resistance training increase both lymphocyte activation and proliferation. We quantified resistance exercise-induced changes in the activation state of CD4+ T lymphocytes via surface protein expression and using a medically relevant model of infection (HIV-1). Using a randomized cross-over design,10 untrained subjects completed a control and exercise session. The control session consisted of 30-min seated rest while the exercise session entailed 3 sets × 10 repetitions of back squat,leg press,and leg extensions at 70{\%} 1-RM with 2-min rest between each set. Venous blood samples were obtained pre/post each session. CD4+ T lymphocytes were isolated from whole blood by negative selection. Expression of activation markers (CD69 {\&} CD25) in both nonstimulated and stimulated (costimulation through CD3+ CD28) cells were assessed by flow cytometry. Resistance exercised-induced effects on intracellular activation was further evaluated via in vitro infection with HIV-1. Nonstimulated CD4+ T lymphocytes obtained postexercise exhibited elevated CD25 expression following 24 h in culture. Enhanced HIV-1 replication was observed in cells obtained postexercise. Our results demonstrate that an acute bout of resistance exercise increases the activation state of CD4+ T lymphocytes and results in a greater susceptibility to HIV-1 infection in vitro. These findings offer further evidence that exercise induces activation of T lymphocytes and provides a foundation for the use of medically relevant pathogens as indirect measures of intracellular activation. View Publication

Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons,cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids,T lymphocytes,and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly,nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac,neurological,or other disease associations. Overall,PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling,and variant-preferred healthy control lines were identified for specific disease settings. View Publication

A rapidly increasing number of reports on dysregulated long intergenic non-coding RNA (lincRNA) expression across numerous types of cancers indicates that aberrant lincRNA expression may be a major contributor to tumorigenesis. Marek's disease (MD) is a T cell lymphoma of chickens induced by Marek's disease virus (MDV). Although we have investigated the roles of lincRNAs in bursa tissue of MDV-infected chickens in previous studies,the molecular mechanisms of lincRNA functions in T cells remain poorly understood. In the present study,Linc-GALMD1 was identified from CD4+ T cells and MSB1 cells,and its expression was significantly downregulated in MD-resistant line of birds in response to MDV challenge. Furthermore,loss-of-function experiments indicated that linc-GALMD1 significantly affected the expression of 290 genes in trans. Through integrated analysis of differentially expressed genes (DEGs) induced by MDV and linc-GALMD1,we found that IGLL1 gene expression levels had a positive correlation with the degree of MD infection and could potentially serve as an indicator for clinical diagnosis of MD. Moreover,an interaction between MDV and linc-GALMD1 was also observed. Accordingly,chicken embryonic fibroblast cells were inoculated with MDV with and without the linc-GALMD1 knockdown,and the data showed that linc-GALMD1 could repress MDV gene expression during the course of MDV infection. These findings uncovered a role of linc-GALMD1 as a viral gene regulator and suggested a function of linc-GALMD1 contributing to tumor suppression by coordinating expression of MDV genes and tumor-related genes and regulating immune responses to MDV infection. View Publication

Exosomes are small extracellular vesicles (sEVs),playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here,we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions,including cytostatic,heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance,melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study,we concluded that (i) molecular patterns of tumour-derived sEVs,dictated by the microenvironmental conditions,resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response,with a potential influence on treatment efficacy. View Publication

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA,but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages,which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™,iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints,and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery,mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1,2 and 4weeks]). 4 weeks after injection,mice were imaged by MRI,and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS,CD206,F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory,homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively,and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs,although some ferumoxytol label was detected in proximity to MSCs in macrophages,likely due to phagocytosis of apoptotic MSCs,increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time,we show that MSC-treated mice had increased ratios of {\%}CD206+: {\%}F4/80+ (homeostatic macrophages) (p{\textless}0.05),and decreased ratios of {\%}iNOS+: {\%}F4/80+ macrophages (p{\textless}0.01),supporting our hypothesis that MSCs may modulate synovial inflammation. View Publication

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme,thereby correcting the biochemical defect and halting disease progression. Here,we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels,maintain long-term repopulation and multi-lineage differentiation potential,and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders. View Publication

Foxp3+ Regulatory T cells (Tregs) are pivotal for the maintenance of tolerance. Alterations in their number and/or function have been proposed to occur in the autoimmune-prone non-obese diabetic (NOD) mouse. Comparing the frequencies and absolute numbers of CD4+Foxp3+CD25+ Tregs among 4 to 6-week old NOD,B6,and BALB/c mice,we observed differences in counts and Foxp3 expression in Tregs from secondary lymphoid organs,but not in the thymus. Upon TCR and IL-2 stimulation,NOD Tregs showed lower responses than Tregs from B6 and BALB/c mice. Indeed,NOD Tregs responded with less proliferation and with smaller increments in the expression of CD25,LAP-1,CD39,PD-1,PD-L1,and LAG-3,when in vitro cultured for 3 days with anti-CD3/CD28 in the absence or presence of IL-2,Tregs from NOD mice showed to be highly dependent on IL-2 to maintain Foxp3 expression. Moreover,NOD Tregs become producers of IL-17 and INF-gamma more easily than Tregs from the other strains. In addition,NOD Tregs showed lower responsiveness to IL-2,with significantly reduced levels of pSTAT5,even at high IL-2 doses,with respect to B6 and BALB/c Tregs. Interestingly,NOD Tregs exhibit differences in the expression of SOCS3,GRAIL,and OTUB1 when compared with Tregs from B6 and BALB/c mice. Both,at steady state conditions and also after activation,Tregs from NOD mice showed increased levels of OTUB1 and low levels of GRAIL. In addition,NOD Tregs had differences in the expression of ubiquitin related molecules that play a role in the maintenance of Foxp3 cellular pools. Indeed,significantly higher STUB1/USP7 ratios were detected in NOD Tregs,both at basal conditions and after stimulation,compared to in B6 and BALB/c Tregs. Moreover,the addition of a proteasome inhibitor to cell cultures,conferred NOD Tregs the ability to retain Foxp3 expression. Herein,we provide evidence indicating a differential expression of SOCS3,GRAIL,and STUB1/USP7 in Tregs from NOD mice,factors known to be involved in IL-2R signaling and to affect Foxp3 stability. These findings add to the current knowledge of the immunobiology of Tregs and may be related to the known insufficiency of Tregs from NOD mice to maintain self-tolerance. View Publication