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Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live,but not killed,virulent MTB. Additionally,we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live,virulent MTB,and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases,and by knockdown of crucial transcriptional regulators,we demonstrate that protein kinase-A (PKA),CREB,and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis,we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live,virulent MTB to subvert the immune system to survive and replicate in the host. View Publication

Canonically,immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE),IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs),a type of cell of the immune system linked to viral defense,which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE,followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses. View Publication

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse,we recently identified the β cell secretory granule protein,chromogranin A (ChgA),as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis,we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast,in NOD.ChgA(+/+) mice,a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice. View Publication

BACKGROUND Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore,the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4(+) T cells-mediated allograft rejection. Hence,we focus on optimizing hESCs for clinic application through gene modification. RESULTS Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA (-/-) hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA (-/-) hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA (-/-) hESCs were powerless in IFN-$\$ expression of HLA II. CONCLUSION We generated HLA II defected hESCs via deleting CIITA,a master regulator of constitutive and IFN-$\$ expression of HLA II genes. CIITA (-/-) hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA (-/-) hESCs-derived cells could be used in cell therapy (e.g.,T cells and DCs) and escape the attack of receptors' CD4(+) T cells,which are the main effector cells of cellular immunity in allograft. 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

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. 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

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT),underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest,but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs,but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement,while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally,adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL. View Publication

T-cell genome engineering holds great promise for cell-based therapies for cancer,HIV,primary immune deficiencies,and autoimmune diseases,but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types� View Publication

Regulatory T cells (Tregs) are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments,their presence is related to a poor prognosis,and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study,we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs,restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70,an increase in the Foxp3 methylation status and,ultimately,the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions,suggesting its possible use in the design of anticancer vaccines. View Publication

BACKGROUND Haemolytic infection lyses red blood cells,releasing haemoglobin (Hb) into the plasma. Although recent studies showed that immune cells recognize redox-active cytotoxic extracellular Hb (metHb) bound to pathogen-associated molecular patterns (PAMPs),currently available information is limited to experiments performed in defined conditions using single cell lines. Therefore,a systemic approach targeting primary whole blood cells is required to better understand the cellular immune defence against metHb and PAMPs,when under a haemolytic infection. METHODS We investigated how human white blood cells,including neutrophils,respond to metHb and lipoteichoic acid (LTA) by measuring reactive oxygen species (ROS),signalling mediators (ERK and p38),NF-κB,cytokines,elastase secretion and cell activation markers. FINDINGS metHb activates NF-κB in TLR2-expressing HEK293 cells but not in normal or TLR9-expressing HEK293 cells. Treatment of isolated neutrophils with metHb increased production of ROS and expressions of IL-8,TNFα,and CD11b,which were further enhanced by metHb + LTA complex. While LTA stimulated the survival of neutrophils,it caused apoptotic cell death when complexed with metHb. The activation of neutrophils by metHb + LTA was subdued by the presence of other types of white blood cells. INTERPRETATION metHb and metHb + LTA complex are ligands of TLR2,inducing an unconventional TLR signalling pathway. Neutrophils are a highly sensitive cell type to metHb + LTA complex. During a haemolytic infection,white blood cells in the vicinity crosstalk to modulate neutrophil TLR-signalling induced by metHb and LTA. View Publication

PURPOSE We conducted an open-label,single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses,respectively. Overall survival (OS),followed as a part of the safety evaluation,was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval,followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months,whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26,95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33,95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS In patients with mCRPC,the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells. View Publication