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G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts,which eventually blocks signal propagation and often induces receptor internalization. However,there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2,GRK3,GRK5,GRK6) simultaneously,but we have very limited knowledge about their interplay in primary immune cells. In particular,we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type,and (b) one specific GPCR between several immune cell subsets. To address this issue,we generated mouse models of single,combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils,T cells,B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes,many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands,whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types,and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly,we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis,which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together,our findings demonstrate the complexity of GRK functions in immune cells,which go beyond GPCR desensitization in specific leukocyte types. Furthermore,they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset. View Publication

Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here,we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels,we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis,phospholipid composition,and inner leaflet surface charge,thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together,our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration. The biomechanical mechanisms enabling the invasive growth of brain tumors remain opaque. Here,Junqueira Alves et al. reveal that the guidance receptor Plexin-B2 controls membrane tension,facilitating confined migration of brain tumor cells. View Publication

Mycobacterium tuberculosis ( Mtb ),the causative agent of tuberculosis (TB),is the most common coinfection among people living with HIV-1. This coinfection is associated with accelerated HIV-1 disease progression and reduced survival. However,the impact of the HIV-1/TB coinfection on HIV-1 replication and latency in CD4 + T cells remains poorly studied. Using the acellular fraction of tuberculous pleural effusion (TB-PE),we investigated whether viral replication and HIV-1 latency in CD4 + T cells are affected by a TB-associated microenvironment. Our results revealed that TB-PE impaired T cell receptor-dependent cell activation and decreased HIV-1 replication in CD4 + T cells. Moreover,this immunosuppressive TB microenvironment promoted viral latency and inhibited HIV-1 reactivation. This study indicates that the TB-induced immune response may contribute to the persistence of the viral reservoir by silencing HIV-1 expression,allowing the virus to persist undetected by the immune system,and increasing the size of the latent HIV-1 reservoir. Subject areas: Immunology,Virology View Publication

Both antitumor and protumor property of mesenchymal stem cells (MSCs) have been demonstrated. We hypothesize that this contradiction is due to the heterogeneity of MSC subsets and that extracellular vesicles (EVs) from distinct MSC subsets can transfer the corresponding antitumor activities. Here we evaluated the antitumor activities of two subsets of adipose-derived mesenchymal stem cells (ADSCs) and ADSC-derived EVs (ADSC-EVs) in immunocompetent syngeneic mouse models of breast cancer. We identified CD90high and CD90low ADSC subsets and demonstrated that CD90high ADSCs could be converted into CD90low ADSCs by stimulation with LPS. CD90low ADSCs and its derived EVs significantly inhibited tumor growth in tumor-bearing mice. Benefit of tumor control were associated with decreased tumor cell proliferation and migration,and enhanced tumor cell apoptosis mediated by ADSC-EVs. Antioncogenic miRNA-16-5p loaded CD90low ADSC-EVs further significantly enhanced antitumor activities. Taken together,this study represents the first attempt to apply our newly identified antitumor ADSCs and its derived EVs in preclinical treatment of breast cancer. This study also provides the evidence that EVs can serve as a novel and effective therapeutics or drug delivery vesicle. This new therapeutic approach could be potentially applicable to breast cancer and many other types of cancer. View Publication

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells,but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall,we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay,the neural colony forming cell assay (N-CFCA),and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis,with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover,the greatest variability occurred in the rostral portion of the lateral ventricles,thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly,LRC numbers were significantly reduced (1186 +/- 188,7 month chase) in comparison to both total colonies and neurospheres. Moreover,approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+),and proliferate upon transfer to culture,it is unclear whether this technique selectively detects endogenous NSCs. Overall,caution should be taken with the interpretation and employment of all these techniques. View Publication

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study,we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab,a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines,cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling,as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines,as indicated by the significant decrease in SFE and the loss of serial transplantability,following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here,we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression,thereby representing a critical survival pathway of tumor-initiating cells. View Publication

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy,given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens,including prostate stem-cell antigen (PSCA),are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial,it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with on-target off-tumor" activity. Here View Publication

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling,quiescent progenitors are present in most samples. In the current study,(3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0),G(1),and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results,with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly,after 72 hours in serum-free culture with or without Steel factor (SF),Flt-3 ligand (FL),and interleukin-3 (IL-3),most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours,1.2% to 37%,and 0% to 7.6% in cultures without and with growth factors [GFs],respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF,FL,IL-3,and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition,3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary,quiescent leukemic cells,including NOD/SL-ICs,are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs. View Publication

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine,but not human cells,and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models,NAb binding to porcine endothelial cells will likely induce complement activation,lysis,and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts,either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis,direct xenogeneic NK lysis,NAb-dependent ADCC,and adhesion of human NK cells under shear stress. Homologous recombination,panning,and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line,PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However,direct xenogeneic lysis of PED2*3.51,mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92,was not reduced. Furthermore,adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion,removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC,but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity,indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells. View Publication

Diffuse alveolar hemorrhage (DAH),although rare,is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans,although increasingly neutrophils,NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model,we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress,namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model,with accompanying reduction in IFN-driven genes and pathology. Lastly,coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH. View Publication

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs,focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations,gene expression,and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma,including severe type 2 inflammation,airway fibrosis,and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages,especially M2a and M2c. Furthermore,MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation,that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes,which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts,which were also suppressed by MSC treatment. In conclusion,intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management. View Publication