技术资料

B-cell functions in antitumor immunity are not well understood. In this study,we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag),D5 mouse melanoma,or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments,spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors),IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells. View Publication

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu,Env,and Nef to down-modulate its primary CD4 receptor from the cell surface,and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation,however,is currently not well understood. In the present study,we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu,env,and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison,Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences,Nef inhibited superinfection more efficiently than Vpu and Env. Notably,nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus,maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally,we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly,one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production. View Publication

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV),another member of the same subfamily. hMPV causes respiratory tract illnesses that,similar to human RSV,occur predominantly during the winter months and have symptoms that range from mild to severe cough,bronchiolitis,and pneumonia. Like RSV,the hMPV virus can be subdivided into two genetic subgroups,A and B. With RSV,a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups,this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV. View Publication

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor,E.G7-OVA,was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice,although both routes primed OVA-specific immune responses,which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice,suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA,but few were detected in primary ocular tumors. Nevertheless,growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice,and CD8(+) T cell numbers were increased within eyes,suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However,CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus,CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye. View Publication

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM,BCR cross-linking led to a quick burst of Syk,ERK1/2,and p38 signaling. In contrast,IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders,such as autoimmunity and cancer. View Publication

Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4,CD25,and the transcription factor,FoxP3. However,these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4(+) T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3(+),including those that express low levels or no CD25. A combination of CD4,CD25,and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact,cells separated based solely on CD4 and CD127 expression were anergic and,although representing at least three times the number of cells (including both CD25(+)CD4(+) and CD25(-)CD4(+) T cell subsets),were as suppressive as the classic" CD4(+)CD25(hi) T reg cell subset. Finally� View Publication

We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab,suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore,cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9,8,and 3. The mitochondrial damage and cell death can be prevented by iron supplementation,but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation,but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av,a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma. View Publication

Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell,followed by actin polymerization,and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here,by using the YTS NK cell line as a model,CD28 is shown to be an activating receptor. It signals cytotoxicity in a process dependent on phosphoinositide-3 kinase activation,leading to sustained extracellular signal-regulated kinase 2 (ERK2) phosphorylation. ERK and phospho-ERK localize to microtubule filaments. Neither conjugation with targets nor actin polymerization is affected by blocking ERK2 activation. However,both polarization of the MTOC and cytolytic granules to the synaptic region and NK cell cytotoxicity are strongly reduced by blocking ERK2 activation. A role for the CD28/CD80 interaction in cytotoxicity of human peripheral NK cells also was established. By contrast,lymphocyte function-associated antigen 1 (LFA-1) ligation transduces only a transient ERK2 activation and fails to induce killing in YTS cells. Thus,in YTS cells,a CD28 signal is used to polarize the MTOC and cytolytic granules to the NK cell immune synapse by stimulating sustained ERK2 activation. View Publication

Respiratory failure during Pneumocystis pneumonia is mainly a consequence of exaggerated inflammatory responses to the organism. Dendritic cells (DCs) are the most potent APCs in the lung and are key to the regulation of innate and adaptive immune responses. However,their participation in the inflammatory response directed against Pneumocystis infection has not been fully elucidated. Therefore,we studied the role of Pneumocystis carinii,as well as Saccharomyces cerevisiae,cell wall-derived beta-glucans,in DC costimulatory molecule expression. We further studied the impact of beta-glucans on subsequent T cell activation. Because cytokine secretion by DCs has recently been shown to be regulated by Fas ligand (FasL),its role in beta-glucan activation of DCs was also investigated. beta-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by beta-glucans elicits T cell activation and polarization into a Th1 patterned response,but with the conspicuous absence of IL-12. These observations differed from LPS-driven T cell polarization,suggesting that beta-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1beta and TNF-alpha secretion by beta-glucan-stimulated DCs was partially regulated by Fas-FasL. This suggests that dysregulation of FasL could further enhance exuberant and prolonged cytokine production by DCs following DC-T cell interactions,further promoting lung inflammation typical of Pneumocystis pneumonia. View Publication

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However,the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study,we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow,in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells,NK progenitors,and NKG2A-negative NK cells),and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore,we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally,we show that acetylated histones are associated with the CpG-rich region in NKG2A positive,but not negative,cell lines,and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription,but cotreatment with both drugs resulted in a significantly greater induction,suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells. View Publication

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands,C3d/iC3b,EBV-gp350,and CD23,a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha,a multifunctional cytokine important in the innate immune system,has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study,we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition,we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b,EBV-gp350,and CD23. Finally,we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus,IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction. View Publication

Langerhans cells (LC) are a unique subset of dendritic cells (DC),present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses,we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types,stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally,CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together,our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system. View Publication