技术资料

The generation and maintenance of virus-specific CD4(+) T cells in humans are not well understood. We used short in vitro stimulation assays followed by intracellular cytokine staining to characterize the timing,magnitude,and Ag specificity of CD4(+) T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4(+) T cells were readily detected at presentation with acute infectious mononucleosis and declined rapidly thereafter. Responses to BZLF-1,BMLF-1,and Epstein-Barr nuclear Ag-3A were more commonly detected than responses to Epstein-Barr nuclear Ag-1. Concurrent analyses of BZLF-1-specific CD4(+) and CD8(+) T cells revealed differences in the expansion,specificity,and stability of CD4(+) and CD8(+) T cell-mediated responses over time. Peripheral blood EBV load directly correlated with the frequency of EBV-specific CD4(+) T cell responses at presentation and over time,suggesting that EBV-specific CD4(+) T cell responses are Ag-driven. View Publication

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However,because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor,we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells,HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore,a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5,demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly,high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However,neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing. View Publication

In this study,we examined the effects of isoform-specific functional inhibitors of lysophosphatidic acid acyltransferase (LPAAT),which converts lysophosphatidic acid to phosphatidic acid,on multiple myeloma (MM) cell growth and survival. The LPAAT-beta inhibitors CT-32176,CT-32458,and CT-32615 induced textgreater95% growth inhibition (P textless 0.01) in MM.1S,U266,and RPMI8226 MM cell lines,as well as MM cells from patients (IC(50),50-200 nM). We further characterized this LPAAT-beta inhibitory effect using CT-32615,the most potent inhibitor of MM cell growth. CT-32615 triggered apoptosis in MM cells via caspase-8,caspase-3,caspase-7,and poly (ADP-ribose) polymerase cleavage. Neither interleukin 6 nor insulin-like growth factor I inhibited CT-32615-induced apoptosis. Dexamethasone and immunomodulatory derivatives of thalidomide (IMiDs),but not proteasome inhibitor PS-341,augmented MM cell apoptosis triggered by LPAAT-beta inhibitors. CT-32615-induced apoptosis was associated with phosphorylation of p53 and c-Jun NH(2)-terminal kinase (JNK); conversely,JNK inhibitor SP600125 and dominant-negative JNK inhibited CT-32615-induced apoptosis. Importantly,CT-32615 inhibited tumor necrosis factor-alpha-triggered nuclear factor-kappaB activation but did not affect either tumor necrosis factor-alpha-induced p38 mitogen-activated protein kinase phosphorylation or interleukin 6-triggered signal transducers and activators of transcription 3 phosphorylation. Finally,although binding of MM cells to bone marrow stromal cells augments MM cell growth and protects against dexamethasone-induced apoptosis,CT-32615 induced apoptosis even of adherent MM cells. Our data therefore demonstrate for the first time that inhibiting LPAAT-beta induces cytotoxicity in MM cells in the bone marrow milieu,providing the framework for clinical trials of these novel agents in MM. View Publication

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Malignant pleural mesothelioma is an uncommon tumor largely confined to the thoracic cavity,which is resistant to conventional therapies,therefore prompting an intensive search for effective treatment alternatives. This study focuses on dendritic cell (DC) vaccination for malignant pleural mesothelioma and evaluates the in vitro efficacy of antigen-loaded DC-based vaccines for the induction of major histocompatibility complex Class I-restricted antimesothelioma cytotoxic T lymphocyte responses. The source of tumor-associated antigens for HLA-A2(+) DCs from healthy donors was apoptotic HLA-A2(-) mesothelioma cells either lacking or expressing heat shock protein 70 according to whether tumor cells were heat shocked or not before ultraviolet-mediated apoptosis. Our results show that both apoptotic preparations were equivalent regarding the responsiveness of DCs to combined treatment with tumor necrosis factor-alpha and poly(inosinic-cytidylic) acid,as determined by similar increased expression of costimulatory molecules and interleukin-12 production. However,only DCs loaded with apoptotic heat shock protein 70-expressing cells were found to be potent in vitro inducers of cytotoxic T lymphocyte activity against HLA-A2(+) mesothelioma cells. Such elicited cytotoxic T lymphocytes also exhibit cytotoxic activity against an HLA-A2(+) melanoma cell line,suggesting recognition of shared antigens. These findings therefore carry the potential of offering an alternative,promising approach for the therapy of patients with malignant pleural mesothelioma. View Publication

Interleukin-10 (IL-10) has potent immunoregulatory effects on the maturation and the antigen-presenting cell (APC) function of dendritic cells (DCs). The molecular basis underlying these effects in DCs,however,is ill defined. It is well established that the transcription factor NF-kappaB is a key regulator of DC development,maturation,and APC function. This study was initiated to determine the effects of IL-10 on the NF-kappaB signaling pathway in immature DCs. IL-10 pretreatment of myeloid DCs cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-kappaB after anti-CD40 antibody or lipopolysaccharide (LPS) stimulation. Furthermore,inhibited NF-kappaB activation was characterized by reduced degradation,phosphorylation,or both of IkappaBalpha and IkappaBepsilon but not IkappaBbeta and by reduced phosphorylation of Ser536,located in the trans-activation domain of p65. Notably,IL-10-mediated inhibition of NF-kappaB coincided with suppressed IkappaB kinase (IKK) activity in vitro. Furthermore,IL-10 blocked inducible Akt phosphorylation,and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed the activation of Akt,IKK,and NF-kappaB. These findings demonstrate that IL-10 targets IKK activation in immature DCs and that suppressing the PI3K pathway in part mediates blockade of the pathway. View Publication

Using whole-cell patch-clamp,fluorescence microscopy and flow cytometry,we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD(+)CD27(+) memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca(2+)-activated intermediate-conductance IKCa1 channel when quiescent,and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast,quiescent class-switched memory B cells express high levels of Kv1.3 ( approximately 2000 channels/cell) and maintain their Kv1.3(high) expression after activation. Consistent with their channel phenotypes,proliferation of naive and IgD(+)CD27(+) memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin,whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore,specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders. View Publication

Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor,c-mpl,activates multiple pathways including signal transducer and activator of transcription (STAT)3,STAT5,phosphoinositide 3-kinase-Akt,and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here,we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl,and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)-derived megakaryocyte culture. Consistent with this result,we found that in both BM and spleen,Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice. In addition,Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3,STAT5,Akt,and MAPK signaling pathways in CD41+ megakaryocytes. Furthermore,the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast,the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to,but is not essential for,inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus,Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo. View Publication

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection,but do not correlate with control of viremia in chronic infection,suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here,we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection,but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies,and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection,but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions. View Publication

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance,we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk,peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless,XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance. View Publication

Primary sclerosing cholangitis (PSC),a chronic inflammatory liver disease characterized by progressive bile duct destruction,develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman,R.W. 1991. Gut. 32:1433-1435). However,the liver and bowel inflammation are rarely concomitant,and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut,but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant,A.J.,P.F. Lalor,M. Salmi,S. Jalkanen,and D.H. Adams. 2002. Lancet. 359:150-157). In support of this,we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD. View Publication

The leukocyte activation marker CD69 is a novel regulator of the immune response,modulating the production of cytokines including transforming growth factor-beta (TGF-beta). We have generated an antimurine CD69 monoclonal antibody (mAb),CD69.2.2,which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell-dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69(-/-) mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-beta production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor-independent manner,resulting in a substantial increase in both NK-cell cytolytic activity and interferon gamma (IFNgamma) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis. View Publication