技术资料

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition,vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy. View Publication

Lymphocyte traffic is required to maintain homeostasis and perform appropriate immunological reactions. To migrate into inflamed tissues,lymphocytes must acquire spatial and functional asymmetries. Mitochondria are highly dynamic organelles that distribute in the cytoplasm to meet specific cellular needs,but whether this is essential to lymphocyte functions is unknown. We show that mitochondria specifically concentrate at the uropod during lymphocyte migration by a process involving rearrangements of their shape. Mitochondrial fission facilitates relocation of the organelles and promotes lymphocyte chemotaxis,whereas mitochondrial fusion inhibits both processes. Our data substantiate a new role for mitochondrial dynamics and suggest that mitochondria redistribution is required to regulate the motor of migrating cells. View Publication

Interferon regulatory factor-1 (IRF-1) is an important transcription factor that mediates interferon-gamma (IFN-gamma)-induced cell-signaling events. In this study,we examined whether 17beta-estradiol alters IRF-1 in splenic lymphocytes,in view of the immunomodulatory effects of this natural female sex hormone including its ability to alter IFN-gamma levels. We find that IRF-1 expression is markedly downregulated in splenocytes or purified T-cells from estrogen-treated mice at all time points studied when compared with their placebo counterparts. This decrease in IRF-1 in splenocytes from estrogen-treated mice is neither due to upregulation of IRF-1-interfering proteins (nucleophosmin or signal transducer and activator of transcription (STAT)-5) nor due to alternatively spliced IRF-1 mRNA. Given that IFN-gamma is a potent inducer of IRF-1,direct addition of recombinant IFN-gamma to splenocytes from either wild-type or IFN-gamma-knockout mice,or the addition of recombinant IFN-gamma to purified T-cells,was expected to stimulate IRF-1 expression. However,robust expression of IRF-1 in cells from estrogen-treated mice was not seen,unlike what was observed in cells from placebo-treated mice. Diminished IFN-gamma induction of IRF-1 in cells from estrogen-treated mice was noticed despite comparable phosphorylated STAT-1 activation. These studies are the first to show that estrogen regulates IFN-gamma-inducible IRF-1 in lymphoid cells,a finding that may have implications to IFN-gamma-regulated immune and vascular diseases. View Publication

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins,as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells,by enzyme-linked immunosorbent assay,immunoprecipitation followed by Western blotting,and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system,and that all forms of the recombinant proteins have in vitro functional activity. Furthermore,we find that in addition to the homodimers of IL-17F and IL-17A,activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses. View Publication

Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%),the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione,increases oxidized intracellular glutathione),whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore,we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding,we suggest,may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo,the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus,we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses,particularly after several days in culture. View Publication

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication

Hyper-IgM (HIGM) syndromes are primary immunodeficiencies characterized by defects of class switch recombination and somatic hypermutation. HIGM patients who carry mutations in the CD40-ligand (CD40L) gene expressed by CD4(+) T cells suffer from recurrent infections and often develop autoimmune disorders. To investigate the impact of CD40L-CD40 interactions on human B cell tolerance,we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from three CD40L-deficient patients. Antibody characteristics and reactivity from CD40L-deficient new emigrant B cells were similar to those from healthy donors,suggesting that CD40L-CD40 interactions do not regulate central B cell tolerance. In contrast,mature naive B cells from CD40L-deficient patients expressed a high proportion of autoreactive antibodies,including antinuclear antibodies. Thus,CD40L-CD40 interactions are essential for peripheral B cell tolerance. In addition,a patient with the bare lymphocyte syndrome who could not express MHC class II molecules failed to counterselect autoreactive mature naive B cells,suggesting that peripheral B cell tolerance also depends on major histocompatibility complex (MHC) class II-T cell receptor (TCR) interactions. The decreased frequency of MHC class II-restricted CD4(+) regulatory T cells in CD40L-deficient patients suggests that these T cells may mediate peripheral B cell tolerance through CD40L-CD40 and MHC class II-TCR interactions. View Publication

BACKGROUND: Basophils are increasingly utilized as indicators of allergic inflammation and as primary allergic effector cells to study signalling pathways. However,until the present,their enrichment has been time consuming,costly and limited to relatively few specialized laboratories. OBJECTIVE: We have therefore devised a reproducible and rapid method for the purification of human basophils from small quantities of peripheral blood within 1.5 h,which does not require the use of specialized equipment such as elutriators. METHODS: Human basophils were obtained from healthy volunteers undergoing venipuncture. Heparinized or K3-ethylenediaminetetraacetic acid blood samples were first subjected to centrifugation in Hetasep,directly followed by negative selection using immunomagnetic beads. Basophil morphology and purity were assessed by May-Grünwald staining of cytospins. IgE-mediated histamine release was analysed spectrofluorometrically and IL-4 and IL-13 production by quantitative RT-PCR. CD203c and CD63 surface expression was measured using flow cytometry before and after activation with anti-IgE. RESULTS: Using this protocol,basophils were enriched close to homogeneity in most cases with a mean purity of 99.34+/-0.88% (range 97-100%,n=18) and a mean recovery of 75.6 (range 39-100%,n=8). Basophil viability following purification was 99.6+/-0.89% using Trypan blue exclusion. The purification procedure gave rise to basophils with normal functional responses to anti-IgE regarding histamine release as well as IL-4 and IL-13 mRNA expression. Moreover,constitutive cell-surface CD203c/CD63 expressions were not elevated before anti-IgE stimulation. CONCLUSION: The rapidity,simplicity and reproducibility of this method will facilitate the employment of basophils in high-output ex vivo studies. View Publication

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study,we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs,characterized by transcriptional repression of IL-1α and β,IL-1R1,and vascular endothelial growth factor A,and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs,therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated,at least in part,by increases in EPC/CAC proliferation,by decreases in EPC/CAC apoptosis,and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs,and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis,but not anti-neutrophil cytoplasmic Ab-positive vasculitis,showed this pathway to be operational in vivo,with increased IL-1R antagonist,downregulation of vascular endothelial growth factor A,and glomerular and blood vessel decreased capillary density,compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease. View Publication

Although toll-like receptor (TLR) agonists,such as CpG,are used as immunotherapeutic agents in clinical trials for cancer and infectious diseases,their effects are limited and the underlying mechanism(s) that restrains CpG efficacy remains obscure. Here,we show that signal transducer and activator of transcription 3 (Stat3) plays a key role in down-modulating immunostimulatory effects of CpG. In the absence of interleukin-6 (IL-6) and IL-10 induction,CpG directly activates Stat3 within minutes through TLR9. Ablating Stat3 in hematopoietic cells results in rapid activation of innate immunity by CpG,with enhanced production of IFN-gamma,tumor necrosis factor-alpha,IL-12,and activation of macrophages,neutrophils,and natural killer cells marked with Stat1 activation. Innate immune responses induced by CpG in mice with a Stat3-ablated hematopoietic system cause potent antitumor effects,leading to eradication of large (textgreater1 cm) B16 melanoma tumors within 72 h. Moreover,ablating Stat3 in myeloid cells increases CpG-induced dendritic cell maturation,T-cell activation,generation of tumor antigen-specific T cells,and long-lasting antitumor immunity. A critical role of Stat3 in mediating immunosuppression by certain cytokines and growth factors in the tumor microenvironment has been recently documented. By demonstrating direct and rapid activation of Stat3 by TLR agonists,we identify a second level of Stat3-mediated immunosuppression. Our results further suggest that targeting Stat3 can drastically improve CpG-based immunotherapeutic approaches. View Publication

Bacillus anthracis secretes two bipartite toxins,edema toxin (ET) and lethal toxin (LT),which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor,the catalytic subunit of ET,is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here,we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN,a marker of immature DCs,and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3beta,like LPS-matured DCs. Interestingly,cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET. View Publication

Dysregulation of airway inflammation contributes to lung disease in cystic fibrosis (CF). Inflammation is mediated by inflammatory cytokines,including IL-8,which illustrates an increase in biological half-life and proinflammatory activity when bound to glycosaminoglycans (GAGs). The aim of this project was to compare IL-8 and IL-18 for their relative stability,activity,and interaction with GAGs,including chondroitin sulfate,hyaluronic acid,and heparan sulfate,present in high quantities in the lungs of patients with CF. Bronchoalveolar lavage fluid was collected from patients with CF (n = 28),non-CF controls (n = 14),and patients with chronic obstructive pulmonary disease (n = 12). Increased levels of IL-8 and reduced concentrations of IL-18 were detected in bronchial samples obtained from CF individuals. The low level of IL-18 was not a defect in IL-18 production,as the pro- and mature forms of the molecule were expressed and produced by CF epithelial cells and monocytes. There was,however,a marked competition between IL-8 and IL-18 for binding to GAGs. A pronounced loss of IL-18 binding capacity occurred in the presence of IL-8,which displaced IL-18 from these anionic-matrices,rendering the cytokine susceptible to proteolytic degradation by neutrophil elastase. As a biological consequence of IL-18 degradation,reduced levels of IL-2 were secreted by Jurkat T lymphocytes. In conclusion,a novel mechanism has been identified highlighting the potential of IL-8 to determine the fate of other inflammatory molecules,such as IL-18,within the inflammatory milieu of the CF lung. View Publication