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Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study,using a panel of MHC class I- and/or class II-restricted peptides,we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First,we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second,we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC,injected i.v.,leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However,IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v.,a similar pattern of cytokines was observed,but,under these conditions,Th1 cytokines were mainly produced by CD8(+) T cells,while Th2 cytokines were produced by CD4(+) T cells. Thus,this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets. View Publication

Regulatory T cells (T(reg)) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice,depletion of T(reg) by Ab therapy leads to more efficient tumor rejection. T(reg)-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study,we measured the prevalence of T(reg) that coexpress CD4 and CD25 in the PBLs,tumor-infiltrating lymphocytes,and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35),pancreas cancer patients (n = 30),and normal donors (n = 35),the prevalence of T(reg) were 16.6% (SE 1.22),13.2% (SE 1.13),and 8.6% (SE 0.71) of the total CD4(+) cells,respectively. The prevalence of T(reg) were significantly higher in breast cancer patients (p textless 0.01) and pancreas cancer patients (p textless 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes,the T(reg) prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3),respectively. T(reg) constitutively coexpressed CTLA-4 and CD45RO markers,and secreted TGF-beta and IL-10 but did not secrete IFN-gamma. When cocultured with activated CD8(+) cells or CD4(+)25(-) cells,T(reg) potently suppressed their proliferation and secretion of IFN-gamma. We conclude that the prevalence of T(reg) is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These T(reg) may mitigate the immune response against cancer,and may partly explain the poor immune response against tumor Ags. View Publication

Caveolae,specialized flask-shaped lipid rafts on the cell surface,are composed of cholesterol,sphingolipids,and structural proteins termed caveolins; functionally,these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study,we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1,which is usually absent in blood cells,is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by beta-cyclodextrin results in the loss of caveola structure in myeloma cells,as shown by transmission electron microscopy,and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I,growth and survival factors in multiple myeloma,induce caveolin-1 phosphorylation,which is abrogated by pre-treatment with beta-cyclodextrin. Importantly,inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. beta-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore,cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together,this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma. View Publication

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function,the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study,we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg,2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%,but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed,we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins. View Publication

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however,the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects,with a median of 14 individual epitopic regions targeted per person (range,2 to 42),and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median,4,245) among all study participants. However,the number of epitopic regions targeted,the protein subunits recognized,and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals,with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid,sensitive,specific,and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response,even if a comprehensive pan-genome screening approach is applied. View Publication

In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO(+) B7-independent CD4(+) T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known,and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4(+) CD45RO(+) B7-independent T cells. Alloreactive CD4(+) T cell clones and lines were generated against HLA-DR11,DR13,DR4,and DR1 MHC Ags,and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner,and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration,migrated cells were still able to proliferate,but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-gamma restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate,thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse,but this is overcome in an inflammatory milieu. View Publication

Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4(+) cells,activated,phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent,but kinetics of activation was delayed relative to interleukin-2 or interferon-gamma. Activation of STAT1,STAT3,and STAT5 occurred with diverse viral envelope proteins,independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells. View Publication

OBJECTIVE: Dysregulation of the apoptotic mechanisms plays a key role in the accumulation of malignant B-chronic lymphocytic leukemia (B-CLL) cells. The transcription nuclear factor (NF)-kappaB is important for cell survival by regulating the expression of anti-apoptotic genes. Several cytokines can modulate leukemic growth and apoptosis in B-CLL. The aim of this study was to determine whether cytokine-mediated regulation of apoptosis occurs via modulation of NF-kappaB activity in peripheral blood mononuclear cells from B-CLL patients. PATIENTS AND METHODS: We evaluated NF-kappaB activity in peripheral blood mononuclear cells from 15 untreated B-CLL patients and 11 controls in resting conditions and in the presence of phorbol-12-myristate-13-acetate (PMA) and different cytokines by electrophoretic mobility shift assay. Apoptosis was studied by spectrophotometric analysis of DNA fragmentation. RESULTS: We found a constitutive high NF-kappaB activity not induced by PMA in B-CLL patients,in contrast with a normal inducible NF-kappaB activity in controls. In B-CLL cultures,addition of interleukin (IL)-4 and IL-13 increased,whereas transforming growth factor (TGF)-beta reduced NF-kappaB activity compared with unstimulated cultures. Accordingly,IL-4 and IL-13 decreased,whereas TGF-beta increased DNA fragmentation compared with unstimulated cultures. IL-13 and IL-4 production was increased,whereas TGF-beta was reduced in PMA-stimulated and unstimulated cultures from B-CLL patients compared with controls. CONCLUSIONS: B-CLL patients have a constitutive high NF-kappaB activity,which is modulated by cytokines. In particular,TGF-beta displays a pro-apoptotic activity,whereas IL-4 and IL-13 have opposite effects. These cytokine alterations could be responsible for a positive autocrine circuit that maintains leukemic cells in a pre-apoptotic state. View Publication

This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro,in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production,and possibly IgA,specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-beta led to the production of specific IgG3 or IgG1 and/or IgA,respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes. View Publication

Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in resting lymphocytes was recently established,although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study,we used CD4(+) T cells isolated from PPARalpha(-/-) and wild-type mice,as well as cell lines that constitutively express PPARalpha,in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4(+) T cells lacking PPARalpha produce increased levels of IFN-gamma,but significantly lower levels of IL-2 when compared with activated wild-type CD4(+) T cells. Furthermore,we demonstrate that PPARalpha regulates the expression of these cytokines by CD4(+) T cells in part,through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4(+) T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation,and the presence of unliganded PPARalpha effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARalpha with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARalpha. View Publication

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs,or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4(+) T cells. Using CD4(+) T cells from cord blood,we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu,although on resting cells,expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils,CXCR3(+)CD4(+) T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4(+) T cells by comparing its pattern of expression with that of CCR5,another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5,supporting a model of sequential induction of chemokine receptors during CD4(+) T cell differentiation. Taken together,our data show that CXCR3 can be expressed at all stages of CD4(+) T cell activation and differentiation,bridging central function in lymphoid organs and effector function in peripheral tissues. View Publication

Human immunodeficiency virus type 1 (HIV-1) isolates from 20 chronically infected patients who participated in a structured treatment interruption (STI) trial were studied to determine whether viral fitness influences reestablishment of viremia. Viruses derived from individuals who spontaneously controlled viremia had significantly lower in vitro replication capacities than viruses derived from individuals that did not control viremia after interruption of antiretroviral therapy (ART),and replication capacities correlated with pre-ART and post-STI viral set points. Of note,no clinically relevant improvement of viral loads upon STI occurred. Virus isolates from controlling and noncontrolling patients were indistinguishable in terms of coreceptor usage,genetic subtype,and sensitivity to neutralizing antibodies. In contrast,viruses from controlling patients exhibited increased sensitivity to inhibition by chemokines. Sensitivity to inhibition by RANTES correlated strongly with slower replication kinetics of the virus isolates,suggesting a marked dependency of these virus isolates on high coreceptor densities on the target cells. In summary,our data indicate that viral fitness is a driving factor in determining the magnitude of viral rebound and viral set point in chronic HIV-1 infection,and thus fitness should be considered as a parameter influencing the outcome of therapeutic intervention in chronic infection. View Publication