技术资料

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

Resident host microflora condition and prime the immune system. However,systemic and mucosal immune responses to bacteria may be divergent. Our aim was to compare,in vitro,cytokine production by human mononuclear and dendritic cells (DCs) from mesenteric lymph nodes (MLNs) and peripheral blood mononuclear cells (PBMCs) to defined microbial stimuli. Mononuclear cells and DCs isolated from the MLN (n = 10) and peripheral blood (n = 12) of patients with active colitis were incubated in vitro with the probiotic bacteria Lactobacillus salivarius UCC118 or Bifidobacterium infantis 35624 or the pathogenic organism Salmonella typhimurium UK1. Interleukin (IL)-12,tumor necrosis factor (TNF)-alpha,transforming growth factor (TGF)-beta,and IL-10 cytokine levels were quantified by ELISA. PBMCs and PBMC-derived DCs secreted TNF-alpha in response to the Lactobacillus,Bifidobacteria,and Salmonella strains,whereas MLN cells and MLN-derived DCs secreted TNF-alpha only in response to Salmonella challenge. Cells from the systemic compartment secreted IL-12 after coincubation with Salmonella or Lactobacilli,whereas MLN-derived cells produced IL-12 only in response to Salmonella. PBMCs secreted IL-10 in response to the Bifidobacterium strain but not in response to the Lactobacillus or Salmonella strain. However,MLN cells secreted IL-10 in response to Bifidobacteria and Lactobacilli but not in response to Salmonella. In conclusion,commensal bacteria induced regulatory cytokine production by MLN cells,whereas pathogenic bacteria induce T cell helper 1-polarizing cytokines. Commensal-pathogen divergence in cytokine responses is more marked in cells isolated from the mucosal immune system compared with PBMCs. View Publication

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine,dihydrosphingosine,and phytosphingosine have potent antimicrobial activity against oral pathogens. Here,we determined the cytotoxicities of long-chain bases for oral cells,an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this,human oral gingival epithelial (GE) keratinocytes,oral gingival fibroblasts (GF),and dendritic cells (DC) were exposed to 10.0-640.0 μM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin),membrane permeability (uptake of propidium iodide or SYTOX-Green),release of cellular contents (LDH),and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC,which were more susceptible. For DC,0.2-10.0 μM long-chain bases and GML were not cytotoxic; 40.0-80.0 μM long-chain bases,but not GML,were cytotoxic; and 80.0 μM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes,GF,and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens,a finding important to pursuing their future potential in treating periodontal and oral infections. View Publication

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7,a mutant dominant-negative Ikaros protein. In contrast,Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore,Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus,distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137) View Publication

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity,which links a given individual stimulus to a unique activated state. Here,we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells. 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

In early life,a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c(+) DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore,we demonstrated that neonatal DC can efficiently take up,process,and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults,this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo. View Publication

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways. View Publication

BACKGROUND Atopy and viral respiratory tract infections synergistically promote asthma exacerbations. IgE cross-linking inhibits critical virus-induced IFN-α responses of plasmacytoid dendritic cells (pDCs),which can be deficient in patients with allergic asthma. OBJECTIVE We sought to determine whether reducing IgE levels in vivo with omalizumab treatment increases pDC antiviral IFN-α responses in inner-city children with asthma. METHODS PBMCs and pDCs isolated from children with exacerbation-prone asthma before and during omalizumab treatment were stimulated ex vivo with rhinovirus and influenza in the presence or absence of IgE cross-linking. IFN-α levels were measured in supernatants,and mRNA expression of IFN-α pathway genes was determined by using quantitative RT-PCR (qRT-PCR) in cell pellets. FcεRIα protein levels and mRNA expression were measured in unstimulated cells by using flow cytometry and qRT-PCR,respectively. Changes in these outcomes and associations with clinical outcomes were analyzed,and statistical modeling was used to identify risk factors for asthma exacerbations. RESULTS Omalizumab treatment increased rhinovirus- and influenza-induced PBMC and rhinovirus-induced pDC IFN-α responses in the presence of IgE cross-linking and reduced pDC surface FcεRIα expression. Omalizumab-induced reductions in pDC FcεRIα levels were significantly associated with a lower asthma exacerbation rate during the outcome period and correlated with increases in PBMC IFN-α responses. PBMC FcεRIα mRNA expression measured on study entry significantly improved an existing model of exacerbation prediction. CONCLUSIONS These findings indicate that omalizumab treatment augments pDC IFN-α responses and attenuates pDC FcεRIα protein expression and provide evidence that these effects are related. These results support a potential mechanism underlying clinical observations that allergic sensitization is associated with increased susceptibility to virus-induced asthma exacerbations. View Publication

OBJECTIVES: Tolerogenic dendritic cells (tolDCs) constitute a promising experimental treatment for targeting autoreactive T cells in autoimmune diseases,including rheumatoid arthritis (RA). The authors' goal is to bring tolDC therapy for RA to the clinic. Here the authors address key translational issues related to the manufacturing of tolDCs from RA patients with current good manufacturing practice (cGMP)-compliant reagents,the stability of tolDCs,and the selection of suitable quality control markers. METHODS: Human monocyte-derived tolDCs were established from RA patients and healthy controls (HCs) using the immunosuppressive drugs dexamethasone and vitamin D₃,and the cGMP-grade immunomodulator,monophosphoryl lipid A,in the cGMP-compliant medium,CellGroDC. The functionality of tolDCs and tolDC-modulated autologous CD4 T cells was determined by flow cytometry,[³H]thymidine incorporation and ELISA. RESULTS: Clinical-grade tolDCs established from patients with RA exhibit a typical tolerogenic phenotype of reduced costimulatory molecules,low production of proinflammatory cytokines and impaired stimulation of autologous antigen-specific T cells,comparable to HC tolDCs. Toll-like receptor 2 (TLR-2) was highly expressed by tolDCs but not mature DCs. Furthermore,tolDCs suppressed mature DC-induced T cell proliferation,interferon γ and interleukin 17 production,and rendered T cells hyporesponsive to further stimulation. Importantly,tolDCs were phenotypically stable in the absence of immunosuppressive drugs and were refractory to further challenge with proinflammatory mediators. CONCLUSIONS: tolDCs established from patients with RA are comparable to those derived from healthy donors. TLR-2 was identified as an ideal marker for quality control of tolDCs. Potently tolerogenic and highly stable,these tolDCs are a promising cellular therapeutic for tailored immunomodulation in the treatment of RA. View Publication

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1,subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions,including induction of Foxp3(+) regulatory T cells,IgA-secreting B cells,and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo,in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function,namely failure to induce Foxp3(+) regulatory T cells. Furthermore,MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity. View Publication