技术资料

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

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

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling,we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C),R-848,or CpG),in the presence or absence of cytokine (TNF-alpha or IL-3),and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons,the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast,PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation,and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection. View Publication

During an immune response,activated antigen (Ag)-specific T cells condition dendritic cells (DCs) to enhance DC function and survival within the inflamed draining lymph node (LN). It has been difficult to ascertain the role of the tumor necrosis factor (TNF) superfamily member lymphotoxin-alphabeta (LTalphabeta) in this process because signaling through the LTbeta-receptor (LTbetaR) controls multiple aspects of lymphoid tissue organization. To resolve this,we have used an in vivo system where the expression of TNF family ligands is manipulated only on the Ag-specific T cells that interact with and condition Ag-bearing DCs. We report that LTalphabeta is a critical participant required for optimal DC function,independent of its described role in maintaining lymphoid tissue organization. In the absence of LTalphabeta or CD40L on Ag-specific T cells,DC dysfunction could be rescued in vivo via CD40 or LTbetaR stimulation,respectively,suggesting that these two pathways cooperate for optimal DC conditioning. View Publication

Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases,human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs),which are critical components of adaptive immunity,are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here,we report that in contrast to several intracellular bacteria,Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover,Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-alpha),a defect involving the bacterial protein Omp25. Exogenous TNF-alpha addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis,B. suis bvrR and B. suis omp25 mutants,which do not express the Omp25 protein,triggered TNF-alpha production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens,two properties which were dramatically impaired by addition of anti-TNF-alpha antibodies. In light of these data,we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-alpha production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host. View Publication

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3(+) human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1alpha (SDF-1alpha)-stimulated LFA-1-ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly,silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1alpha- and phorbol 12-myristate 13-acetate (PMA)-induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1,whereas adhesion to VCAM-1 was reduced. Thus,PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells,whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4. View Publication

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However,the factors that regulate the magnitude of ADCC are incompletely understood. In this study,we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M,and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting,a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

BAFF plays a central role in B-lineage cell biology; however,the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study,we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation,as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs),excluding bone marrow PCs,express BAFF-R uniformly. In contrast,only tonsillar memory B cells (MB) and PCs,from both tonsil and bone marrow tissues,express BCMA. Furthermore,we show that TACI is expressed by MB cells and PCs,as well as a subpopulation of activated CD27(neg) B cells. In this regard,we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition,we found that TACI expression requires activation of the ERK1/2 pathway,since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC),since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively,our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

Autoimmune pathologies are caused by a breakdown in self-tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self-tolerance in an antigen-specific manner. Studies about tolDC have focused largely on generating stable maturation-resistant DC,but few have fully addressed questions about the antigen-presenting and migratory capacities of these cells,prerequisites for successful immunotherapy. Here,we investigated whether human tolDC,generated with dexamethasone and the active form of vitamin D3,maintained their tolerogenic function upon activation with LPS (LPS-tolDC),while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS-tolDC,but not tolDC,expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore,LPS-tolDC were superior to tolDC in their ability to present type II collagen,a candidate autoantigen in rheumatoid arthritis. tolDC and LPS-tolDC had low stimulatory capacity for allogeneic,naïve T cells and skewed T cell polarization toward an anti-inflammatory phenotype,although LPS-tolDC induced significantly higher levels of IL-10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen-presenting activity in tolDC is important for optimizing their therapeutic potential. View Publication

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally,copy number losses are identified using metaphase cytogenetics,whereas detection of UPD is accomplished by microsatellite and copy number analysis and as such,is not often used clinically. Recently,introduction of single nucleotide polymorphism (SNP) microarrays has allowed for the systematic and sensitive detection of UPD in hematologic malignancies and other cancers. In this study,we have applied 250K SNP array technology to detect previously cryptic chromosomal changes,particularly UPD,in a cohort of 301 patients with myelodysplastic syndromes (MDS),overlap MDS/myeloproliferative disorders (MPD),MPD,and acute myeloid leukemia. We show that UPD is a common chromosomal defect in myeloid malignancies,particularly in chronic myelomonocytic leukemia (CMML; 48%) and MDS/MPD-unclassifiable (38%). Furthermore,we show that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations,including newly identified missense mutations in the proto-oncogene c-Cbl in 7 of 12 patients with UPD11q. Acquired mutations of c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process in a subset of MDS/MPD,including CMML. View Publication