技术资料

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β,a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis,we performed metabolic and functional studies on human alveolar macrophages,human monocyte-derived macrophages,and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2,increased levels of anti-inflammatory IL-10,and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival,demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies. View Publication

Immunotherapy treatments for cancer are becoming increasingly successful,however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy,precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1),MelanA,Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes,and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3),tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses,predict patient response to conventional therapy and direct personalised immunotherapy for patients. View Publication

The lectin galectin-9 may help establish and maintain chronic hepatitis C virus infection. Galectin-9 is elevated in the liver and sera of hepatitis C virus patients,induces apoptosis of hepatitis C virus-specific T cells,and increases inhibitory regulatory T cells. Kupffer cells stain strongly for galectin-9 protein in hepatitis C virus patients. In the current study,we determined stimuli that induce galectin-9 production by monocytes and macrophages in hepatitis C virus infection. With the use of real-time PCR and flow cytometry,we analyzed galectin-9 mRNA and protein from human monocytes cocultured with hepatitis C virus-infected cells or noninfectious hepatitis C virus subgenomic replicon cells. We focused on finding the stimuli for galectin-9 production. Additionally,we measured galectin-9 during monocyte-to-macrophage maturation. Finally,we examined galectin-9 in peripheral monocytes from hepatitis C virus patients using flow cytometry. Galectin-9 mRNA increased 8-fold when primary monocytes were exposed to hepatitis C virus--infected cells. Maximum induction required proximity or contact and did not require IFN-γ or hepatitis C virus virions. Coculture of monocytes with subgenomic replicon cells increased galectin-9 5-fold,and purified exosomes from infected cells stimulated galectin-9 production. Stimulation of monocyte TLR3,-7,and -8 increased galectin-9 production. Differentiation of monocytes to macrophages increased galectin-9,and nonclassic monocytes from hepatitis C virus patients had the highest levels of galectin-9. Hepatitis C virus-infected cells stimulated monocytes to produce galectin-9 in close proximity,possibly,in part,as a result of exosomes and endosomal TLRs. Differentiation of monocytes to macrophages increased galectin-9. Nonclassic monocytes from hepatitis C virus patients express the highest galectin-9 levels,suggesting they may contribute to elevated galectin-9 and adaptive immune inhibition in hepatitis C virus infection. View Publication

HIV-1 infection leads to numerous B cell abnormalities,including hypergammaglobulinemia,nonspecific B cell activation,nonspecific class switching,increased cell turnover,breakage of tolerance,increased immature/transitional B cells,B cell malignancies,as well as a loss of capacity to generate and maintain memory,all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors,which are increased in sera of HIV-1-infected individuals,have been suggested to directly or indirectly contribute to these B cell dysfunctions,and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover,we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes,but not in nonclassical monocytes,which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion. View Publication

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses,the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study,we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression,in this context CTLA-4 blocking Abs had no impact on the response. In contrast,in settings where CTLA-4(+) cells were present as regulators� View Publication

Owing to ongoing recognition of pathogen-associated molecular patterns,immune activation and upregulation of IFN-stimulated genes (ISGs) are sustained in the chronically infected host. Albeit most ISGs are important effectors for containing viral replication,some might exert compensatory immune suppression to limit pathological dysfunctions,although the mechanisms are not fully understood. In this study,we report that the ISG lymphocyte Ag 6 complex,locus E (LY6E) is a negative immune regulator of monocytes. LY6E in monocytes negatively modulated CD14 expression and subsequently dampened the responsiveness to LPS stimulation in vitro. In the setting of chronic HIV infection,the upregulation of LY6E was correlated with reduced CD14 level on monocytes; however,the immunosuppressive effect of LY6E was not adequate to remedy the hyperresponsiveness of activated monocytes. Taken together,the regulatory LY6E pathway in monocytes represents one of negative feedback mechanisms that counterbalance monocyte activation,which might be caused by LPS translocation through the compromised gastrointestinal tract during persistent HIV-1 infection and may serve as a potential target for immune intervention. View Publication

The ontogeny of human Langerhans cells (LCs) remains poorly characterized,in particular the nature of LC precursors and the factors that may drive LC differentiation. Here we report that thymic stromal lymphopoietin (TSLP),a keratinocyte-derived cytokine involved in epithelial inflammation,cooperates with transforming growth factor (TGF)-β for the generation of LCs. We show that primary human blood BDCA-1(+),but not BDCA-3(+),dendritic cells (DCs) stimulated with TSLP and TGF-β harbor a typical CD1a(+)Langerin(+) LC phenotype. Electron microscopy established the presence of Birbeck granules,an intracellular organelle specific to LCs. LC differentiation was not observed from tonsil BDCA-1(+) and BDCA-3(+) subsets. TSLP + TGF-β LCs had a mature phenotype with high surface levels of CD80,CD86,and CD40. They induced a potent CD4(+) T-helper (Th) cell expansion and differentiation into Th2 cells with increased production of tumor necrosis factor-α and interleukin-6 compared with CD34-derived LCs. Our findings establish a novel LC differentiation pathway from BDCA-1(+) blood DCs with potential implications in epithelial inflammation. Therapeutic targeting of TSLP may interfere with tissue LC repopulation from circulating precursors. View Publication

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo,we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood,spleen,and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes,which downregulate GFP expression on differentiation into macrophages in this model,CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages,allowing continued cell tracking during resolution of inflammation. In summary,this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. View Publication

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes,particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore,we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition,we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages,reduced glycolipid storage,and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development. View Publication

As sentinels of the immune system,dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients,Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform,termed ID-VP02,utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx,a SIVmac viral accessory protein,to achieve efficient targeting and transduction of human DCs. In addition,ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here,we describe the characteristics that allow ID-VP02 to specifically transduce human DCs,and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted. View Publication

We describe here the effects of three drugs that are either approved or have the potential for treating multiple sclerosis (MS) patients through the in vitro activities of human natural killer (NK) cells and dendritic cells (DCs). Our results indicate that 1,25(OH)2D3,the biologically active metabolite of vitamin D3,calcipotriol and FTY720 augment IL-2-activated NK cell lysis of K562 and RAJI tumor cell lines as well as immature (i) and mature (m) DCs,with variable efficacies. These results are corroborated with the ability of the drugs to up-regulate the expression of NK cytotoxicity receptors NKp30 and NKp44,as well as NKG2D on the surfaces of NK cells. Also,they down-regulate the expression of the killer inhibitory receptor CD158. The three drugs down-regulate the expression of CCR6 on the surface of iDCs,whereas vitamin D3 and calcipotriol tend to up-regulate the expression of CCR7 on mDCs,suggesting that they may influence the migration of DCs into the lymph nodes. Finally,vitamin D3,calcipotriol and FTY720 enhance NK17/NK1 cell lysis of K562 cells,suggesting that a possible mechanism of action for these drugs is via activating these newly described cells. In conclusion,our results show novel mechanisms of action for vitamin D3,calcipotriol and FTY720 on cells of the innate immune system. View Publication

HIV-1 infected cells are eliminated in infected individuals by a variety of cellular mechanisms,the best characterized of which are cytotoxic T cell and NK cell-mediated killing. An additional antiviral mechanism is antibody-dependent cellular cytotoxicity. Here we use primary CD4(+) T cells infected with the BaL clone of HIV-1 as target cells and autologous NK cells,monocytes,and neutrophils as effector cells,to quantify the cytotoxicity mediated by the different effectors. This was carried out in the presence or absence of HIV-1-specific antiserum to assess antibody-dependent cellular cytotoxicity. We show that at the same effector to target ratio,NK cells and monocytes mediate similar levels of both antibody-dependent and antibody-independent killing of HIV-1-infected T cells. Neutrophils mediated significant antibody-dependent killing of targets,but were less effective than monocytes or NK cells. These data have implications for acquisition and control of HIV-1 in natural infection and in the context of vaccination. View Publication