搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread,requires CD4 and coreceptor expression in target cells,and may also lead to syncytium formation and cell death. Here,we show that in addition to this classical coreceptor-mediated transmission,the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped,captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5-1.0 microm) intracellular vesicles containing 1-14 virions,without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion,we describe a novel mechanism of HIV transmission,activated by the contact of infected and uninfected primary CD4 T cells,by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir. 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

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. 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

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics,we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo,LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3-/- CD4+ T cells,solidifying that LAG-3 controls these processes. Lag3-/- CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation,enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally,enhancement of STAT5 activation,as a result of LAG-3 deficiency,contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. View Publication

PURPOSE Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors,such as prostate cancers. However,targeting MDSCs proved challenging due to their phenotypic heterogeneity. EXPERIMENTAL DESIGN Myeloid cell populations were evaluated using flow cytometry on blood samples,functional assays,and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects,localized and metastatic castration-resistant prostate cancer patients. RESULTS Here,we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3,which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA,thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1,a downstream STAT3 target gene and a potent T-cell inhibitor. CONCLUSIONS Overall,we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression. View Publication

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family,such as NKp46,by ligands expressed on tumour cells. However,it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here,we investigated the early events occurring during T cell-tumour cell interactions,and their impact on NK cell functions. We observed that on co-culture with some melanomas,activated CD4(+) T cells promoted degranulation,and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells,probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+),CD8(+) and resting T cells,which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new,so far,unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. View Publication

BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products,which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities,there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment,and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like,fully defined,serum- and protein-free preservation solution,CryoStor (BioLife Solutions Inc.),with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly,human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard,Normosol-R,5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed,and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC),viability,CD34 and granulocytes by flow cytometry,along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+),colony-forming units (CFU) and granulocytes. Specifically,relative to the FHCRC standard formulation,cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005),a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition,it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins,and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however,these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections,along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO. View Publication

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However,it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs,we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore,the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ,IL-2,IL-6 and TGF-β into coculture supernatants and increased the IL-12p70,IFN-γ,IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly� View Publication

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here,we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes,where they trap T cells by releasing the CCR7 ligand,CCL19,and to inflamed central nervous system,where they locally limit immunopathogenesis through interleukin-10 production,thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment,where proB cells develop,is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition,these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. View Publication