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Many tumors,including Hodgkin's lymphoma,are associated with decreased cellular immunity and elevated levels of prostaglandin E(2) (PGE(2)),a known inhibitor of CD4+ T cell activation,suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE(2) might have on primary human CD4+ T cells,we used a whole genome-based transcriptional approach and show that PGE(2) severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE(2) at an early step of T cell receptor signaling: indeed,PGE(2) stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE(2)-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence,PGE(2)-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27(kip1). Most importantly,CD4+ T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE(2) in T cells from healthy individuals. These data strongly suggest that PGE(2) is an important factor leading to CD4+ T cell impairment observed in Hodgkin's lymphoma. View Publication

To investigate retinal involvement in chronic Chagas' disease,we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency,under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors,all related to the same subfamily of G-protein-coupled receptors. View Publication

Acute coronary syndromes (ACS) are precipitated by a rupture of the atherosclerotic plaque,often at the site of T cell and macrophage infiltration. Here,we show that plaque-infiltrating CD4 T cells effectively kill vascular smooth muscle cells (VSMC). VSMCs sensitive to T cell-mediated killing express the death receptor DR5 (TNF-related apoptosis-inducing ligand [TRAIL] receptor 2),and anti-TRAIL and anti-DR5 antibodies block T cell-mediated apoptosis. CD4 T cells that express TRAIL upon stimulation are expanded in patients with ACS and more effectively induce VSMC apoptosis. Adoptive transfer of plaque-derived CD4 T cells into immunodeficient mice that are engrafted with human atherosclerotic plaque results in apoptosis of VSMCs,which was prevented by coadministration of anti-TRAIL antibody. These data identify that the death pathway is triggered by TRAIL-producing CD4 T cells as a direct mechanism of VSMC apoptosis,a process which may lead to plaque destabilization. View Publication

Perifosine is a synthetic novel alkylphospholipid,a new class of antitumor agents which targets cell membranes and inhibits Akt activation. Here we show that baseline phosphorylation of Akt in multiple myeloma (MM) cells is completely inhibited by perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate] in a time- and dose-dependent fashion,without inhibiting phosphoinositide-dependent protein kinase 1 phosphorylation. Perifosine induces significant cytotoxicity in both MM cell lines and patient MM cells resistant to conventional therapeutic agents. Perifosine does not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous interleukin-6 (IL-6) nor insulinlike growth factor 1 (IGF-1) overcomes Perifosine-induced cytotoxicity. Importantly,Perifosine induces apoptosis even of MM cells adherent to bone marrow stromal cells. Perifosine triggers c-Jun N-terminal kinase (JNK) activation,followed by caspase-8/9 and poly (ADP)-ribose polymerase cleavage. Inhibition of JNK abrogates perifosine-induced cytotoxicity,suggesting that JNK plays an essential role in perifosine-induced apoptosis. Interestingly,phosphorylation of extracellular signal-related kinase (ERK) is increased by perifosine; conversely,MEK inhibitor synergistically enhances Perifosine-induced cytotoxicity in MM cells. Furthermore,perifosine augments dexamethasone,doxorubicin,melphalan,and bortezomib-induced MM cell cytotoxicity. Finally,perifosine demonstrates significant antitumor activity in a human plasmacytoma mouse model,associated with down-regulation of Akt phosphorylation in tumor cells. Taken together,our data provide the rationale for clinical trials of perifosine to improve patient outcome in MM. View Publication

Dendritic cell (DC) maturation state is a key parameter for the issue of DC-T cell cognate interaction,which determines the outcome of T cell activation. Indeed,immature DCs induce tolerance while fully mature DCs generate immunity. Here we show that,in the absence of any deliberate activation signal,DCs freshly isolated from mouse spleen spontaneously produce IL-12 and tumor necrosis factor-alpha and up-regulate co-stimulation molecules,even when directly re-injected into their natural environment. Furthermore,after their isolation,these cells acquire the capacity to induce specific T(h)1 responses in vivo. These results demonstrate that the sole isolation of spleen DCs leads to the full maturation of these cells,which therefore cannot be considered as immature DCs. Moreover,we also show that the kinetics of DC activation do not influence the polarization of T(h) response in vivo challenging the idea that exhausted DCs induce preferentially T(h)2 response. Altogether,these observations should be taken into account in all experiments based on the transfer of ex vivo purified DCs. View Publication

NK cell transcript 4 (NK4),now denoted as IL-32,was originally identified as a transcript whose expression was increased in activated NK cells. It has been very recently demonstrated that NK4 is secreted from several cells upon the stimulation of some inflammatory cytokines such as IL-18,IL-1beta,IFN-gamma and IL-12. Furthermore,NK4 induces production of tumor necrosis factor,macrophage inflammatory protein (MIP)-2 and IL-8 in monocytic cell lines,indicating that this factor would be involved in the inflammatory responses. Based on these findings,NK4 was renamed IL-32. However,the biological activities of IL-32 on other cell types remained undetermined. Furthermore,it was still argued whether IL-32 acts on cells from outside or inside the cells. In this article,we first report that expression of IL-32 was up-regulated in activated T cells and NK cells,and that IL-32beta was the predominantly expressed isoform in activated T cells. IL-32 was specifically expressed in T cells undergoing apoptosis and enforced expression of IL-32-induced apoptosis,whereas its down-regulation rescued the cells from apoptosis in HeLa cells. IL-32 existing in the supernatant would be derived from the cytoplasm of apoptotic cells. These results strongly indicated that IL-32 would be involved in activation-induced cell death in T cells,probably via its intracellular actions. Our present findings expand our understanding of the biological function of IL-32 and argue that IL-32 may act on cells,not only from the outside but also from the inside. View Publication

Chronic myeloid leukemia is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein,a constitutively active tyrosine kinase. In previous studies,we have provided evidence that Bcr/Abl overexpression in leukemic cells increased their susceptibility to NK-mediated lysis by different mechanisms. In the present study,using UT-7/9 cells,a high level Bcr/Abl transfectant of UT-7 cells,we show that the treatment of Bcr/Abl target by imatinib mesylate (IM),a specific Abl tyrosine kinase inhibitor,hampers the formation of the NK/target immunological synapse. The main effect of IM involves an induction of surface GM1 ganglioside on Bcr/Abl transfectants that prevents the redistribution of MHC-related Ag molecules in lipid rafts upon interaction with NK cells. IM also affects cell surface glycosylation of targets,as assessed by binding of specific lectins resulting in the subsequent modulation of their binding to lectin type NK receptor,particularly NKG2D. In addition,we demonstrate that the tyrosine kinase activity repression results in a decrease of MHC-related Ags-A/B and UL-16-binding protein expression on Bcr/Abl transfectants UT-7/9. We show that NKG2D controls the NK-mediated lysis of UT-7/9 cells,and IM treatment inhibits this activating pathway. Taken together,our results show that the high expression of Bcr/Abl in leukemic cells controls the expression of NKG2D receptor ligands and membrane GM1 via a tyrosine kinase-dependent mechanism and that the modulation of these molecules by IM interferes with NK cell recognition and cytolysis of the transfectants. View Publication

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled,but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases,such as systemic lupus erythematosus. Beyond regulation by NFAT proteins,little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site,which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1,Egr-3,and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo,respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1,but not Egr-3,is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely,overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly,upon activation,CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus. View Publication

Natural killer (NK) cells are thought to develop from common lymphoid progenitors in the bone marrow. However,immature thymocytes also retain NK potential. Currently,the contribution of the thymus-dependent pathway in normal steady-state NK-cell development is unknown. Here,we show that TCRgamma genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells,and similar levels are detected in NK cells from TCRbeta,delta double-knockout mice,excluding the possibility of T-cell contamination. NK-cell TCRgamma gene rearrangement is thymus dependent because this rearrangement is undetectable in nude mouse NK cells. These results change the current view of NK-cell development and show that a subset of NK cells develops from immature thymocytes that have rearranged TCRgamma genes. View Publication

Resident host microflora condition and prime the immune system. However,systemic and mucosal immune responses to bacteria may be divergent. Our aim was to compare,in vitro,cytokine production by human mononuclear and dendritic cells (DCs) from mesenteric lymph nodes (MLNs) and peripheral blood mononuclear cells (PBMCs) to defined microbial stimuli. Mononuclear cells and DCs isolated from the MLN (n = 10) and peripheral blood (n = 12) of patients with active colitis were incubated in vitro with the probiotic bacteria Lactobacillus salivarius UCC118 or Bifidobacterium infantis 35624 or the pathogenic organism Salmonella typhimurium UK1. Interleukin (IL)-12,tumor necrosis factor (TNF)-alpha,transforming growth factor (TGF)-beta,and IL-10 cytokine levels were quantified by ELISA. PBMCs and PBMC-derived DCs secreted TNF-alpha in response to the Lactobacillus,Bifidobacteria,and Salmonella strains,whereas MLN cells and MLN-derived DCs secreted TNF-alpha only in response to Salmonella challenge. Cells from the systemic compartment secreted IL-12 after coincubation with Salmonella or Lactobacilli,whereas MLN-derived cells produced IL-12 only in response to Salmonella. PBMCs secreted IL-10 in response to the Bifidobacterium strain but not in response to the Lactobacillus or Salmonella strain. However,MLN cells secreted IL-10 in response to Bifidobacteria and Lactobacilli but not in response to Salmonella. In conclusion,commensal bacteria induced regulatory cytokine production by MLN cells,whereas pathogenic bacteria induce T cell helper 1-polarizing cytokines. Commensal-pathogen divergence in cytokine responses is more marked in cells isolated from the mucosal immune system compared with PBMCs. View Publication

The immune system can act as an extrinsic suppressor of tumors. Therefore,tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here,we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells,and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells,T cells,natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3(-/-) hematopoietic cells,and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell- and NK cell-dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity. View Publication

In recent years,mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However,no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells,whereas they only partially affect the proliferation of activated NK cells. In addition,we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30,NKG2D,and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly,MSCs expressed the known ligands for these activating NK receptors-ULBPs,PVR,and Nectin-2. Moreover,NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation. View Publication