Inhibition of T cell antigen receptor-dependent phosphorylation of CD4 in human immunodeficiency virus type 1 infected cells.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.     

Latent Kaposi's sarcoma-associated herpesvirus infection of monocytes downregulates expression of adaptive immune response costimulatory receptors and proinflammatory cytokines

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is associated with the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. We report the establishment of a monocytic cell line latently infected with KSHV (KSHV-THP-1). We profiled viral and cytokine gene expression in the KSHV-THP-1 cells compared to that in uninfected THP-1 cells and found that several genes involved in the host immune response were downregulated during latent infection, including genes for CD80, CD86, and the cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Thus, KSHV minimizes its immunological signature by suppressing key immune response factors, enabling persistent infection and evasion from host detection.     

Glycosphingolipids of the globo-series are associated with the monocytic lineage of human myeloid cells

Neutral glycosphingolipids (neutral GSLs) of the human myeloid leukemia cell lines ML-2, ML-3, HL-60 and THP-1-0 were metabolically labeled with [3H]galactose and [3H]glucosamine, and analyzed by high-performance liquid chromatography. They were compared with unlabeled neutral GSLs from purified human granulocytes and monocytes. Neutral GSLs were identified by retention times and the structures were further confirmed by degradation with specific exoglycosidases. Two neutral GSLs of the globoseries, globotetraosylceramide and globotriaosylceramide were found in monocytes and the monoblastic leukemia line THP-1-0. The leukemia-derived cell-lines, ML-3 and HL-60, representing successively earlier stages of myeloid differentiation, contained respectively less neutral GSLs of the globoseries and an increasing proportion of (neo)lacto neutral GSLs. Granulocytes and the cell line ML-2 contained almost exclusively neutral GSLs of the (neo)lacto series.     

Effect of fibroblast-derived differentiation inducing factor on the differentiation of human monocytoid and myeloid leukemia cell lines

A fibroblast-derived differentiation inducing factor (F-DIF) purified from medium conditioned by a human fibroblast cell line (WI-26VA4) induced differentiation of human monocytic leukemia cell lines (U-937, THP-1) into cells with macrophage characteristics. F-DIF alone induced the differentiation of ML-1 cells only marginally, but it synergistically increased the differentiation when combined with TNF. Interferon-gamma, tumor necrosis factor, GM-CSF, interleukin-1 and interlukin-4 synergistically enhanced the differentiation of U-937 cells when combined with F-DIF.     

Helicobacter pylori has stimulatory effects on naive T cells

BACKGROUND: Despite an apparently active host response, Helicobacter pylori infection can persist for life. Unexpectedly, T cells from apparently uninfected individuals respond to H. pylori antigen by proliferating. Also, the T-cell proliferative response appears to be less in infected compared with uninfected individuals. MATERIALS AND METHODS: We have investigated the T-cell response of isolated human peripheral blood, naive, and memory CD4+ T cells to H. pylori antigen in infected and uninfected subjects. RESULTS: In agreement with previous findings, the peripheral blood proliferative response was higher in uninfected compared with infected subjects. Interestingly, there was a response in CD4+ CD45RO+ (memory) and CD4+CD45RA+ (naive) subsets. The RO/RA ratio of the response to H. pylori antigen was 0.8-2.1 in both H. pylori-positive and H. pylori-negative subjects, which was similar to that of a known superantigen (2.5 and 2.2 in Helicobacter-positive and -negative subjects, respectively) whereas the RO/RA response ratio to a recall antigen (tetanus toxoid) was 9.8 and 18.7 in Helicobacter-positive and -negative subjects, respectively. Mononuclear cells isolated from cord blood also responded to H. pylori antigen, whereas there was no response to tetanus toxoid. The cord blood response and CD4+ CD45RA+ cell response to H. pylori antigen were inhibited predominantly by anti-HLA-DR and to some extent by anti-HLA-DQ antibodies. Investigation of the response to five different recombinant H. pylori antigens identified two that produced a response in naive T cells. CONCLUSIONS: These data suggest that H. pylori possesses molecules that cause higher than expected proliferation of naive T cells.     

Diagnostic surface expression of SWAP-70 on HIV-1 infected T cells

Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.     

Intracellular overexpression of HIV-1 Nef impairs differentiation and maturation of monocytic precursors towards dendritic cells

Nef functions as an immunosuppressive factor critical for HIV-1 replication, survival and development of AIDS following HIV-1 infection. What effects Nef exerts on differentiation and maturation of monocytes towards dendritic cells (DCs) remains greatly controversial. In this study, we used THP-1 (human monocytic leukemia cell line) as monocytic DC precursors to investigate how overexpression of HIV-1 Nef influences the processes of differentiation and maturation of dendritic cells. In striking contrast to negative controls, our results showed that morphological and phenotypical changes (CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR) occurred on recombinant THP-1 expressing HIV-1 Nef (short for Nef) upon co-stimulation of GM-CSF/IL-4 or GM-CSF/IL-4/TNF-α/ionomycin. Moreover, CD4, CCR5, and CXCR4 were also down-regulated on Nef. It might be hypothesized that Nef prevents superinfection and signal transduction in HIV-1 infected monocytes. Collectively, our study demonstrates that long-lasting expression of Nef at high levels indeed retards differentiation and maturation of dendritic cells in terms of phenotype and morphology. We are hopeful that potentially, stable expression of intracellular Nef in vivo may function as a subtle mode to support long-lasting HIV-1 existence.     

Antibodies enhance the infection of phorbol-ester-differentiated human monocyte-like cells with coxsackievirus B4

Coxsackievirus B4 (CV-B4), in presence of antibodies and through a specific viral receptor CAR and Fcγ receptors II and III, can infect monocytes which results in interferon-α synthesis. The antibody-dependent enhancement of CV-B4 infection in the human monocytic-like THP-1 cell line has been investigated. The preincubation of CV-B4 with human plasma or human polyvalent immunoglobulins enhanced the infection of phorbol–myristate–acetate (PMA)-activated THP-1 cell cultures. CV-B4 replicated in these cells as demonstrated by the intracellular detection of infectious particles, viral protein VP1 (immunofluorescence), positive and negative viral RNA (RT-PCR). The viability of infected and control cell cultures was not different up to 20 days post-infection. Activated cell cultures inoculated with CV-B4 harbored intracellular RNA up to 14 days post-infection and produced IFNα that was detected by intracellular immunofluorescence staining as soon as 4 h post-infection with a maximum at 48 h post-infection and by RT-PCR all along the experiment. Together, these data demonstrate that PMA-activated THP-1 cells can be infected with CV-B4, can produce IFNα as a result of interactions between the virus, antibodies and specific receptors. This cellular model can be used to investigate further the mechanism and the result of the antibody-dependent enhancement of CV-B4 infection.     

Proteomic analysis of the cellular responses induced in uninfected immune cells by cell-expressed X4 HIV-1 envelope

HIV-1 envelope gp120 and gp41 glycoproteins (Env), expressed at the cell surface, induce uninfected CD4 T-cell death, but the molecular mechanisms leading to this demise are still largely unknown. To better understand these events, we analyzed by a proteomic approach the differential protein expression profile of two types of uninfected immune cells after their coculture for 1-3 days with cells that express, or not, Env. First, umbilical cord blood mononuclear cells (UCBMCs) were used to approach the in vivo situation, i.e., blood uninfected naive cells that encounter infected cells. Second, we used the A2.01/CD4.403 T-cell line expressing wild type CXCR4 and a truncated form of CD4 that still undergoes Env-mediated apoptosis, independently of CD4 signaling. After coculture with cells expressing Env, 35 and 39 proteins presenting an altered expression in UCBMCs and the A2.01/CD4.403 T-cell line, respectively, were identified by mass-spectrometry. Whatever the cell type analyzed, the majority of these proteins are involved in degradation processes, redox homeostasis, metabolism and cytoskeleton dynamics, and linked to mitochondrial functions. This study provides new insights into the events that sequentially occur in bystander T lymphocytes after contact with HIV-infected cells and leading, finally, to apoptotic cell death.     

Pathogenic hantaviruses elicit different immunoreactions in THP-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells

Hantaviruses cause two important human illnesses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Both syndromes are believed to be immune-mediated diseases. Monocytes/macrophages are thought to be the main target cells for hantaviruses and important sources of and targets for cytokines/chemokines secretion. THP-1 cells have been used extensively as models for primary monocytes in biocompatibility research. The aim of our study was to determine if hantaviruses induce the same immunoreactions in THP-1 cells and primary monocytes/ macrophages and might therefore be suitable for immune studies of hantaviral infections. For that purpose we compared various cytokines/chemokines and their receptors in THP-1 cell line and primary monocytes/macrophages. Infected primary monocytes/macrophages induced mostly beta-chemokines and their receptors. In contrast, THP-1 cells, expressed receptors for CXC chemokines. Surprisingly, infected macrophages underwent morphological changes toward dendritic-like cells and increased expression of co-stimulatory molecules: CD40, CD80, CD83 and CD86. Our data indicate that THP-1 cells are not ideal for in vitro research of the immunopathogenesis of hantaviruses in humans. Further, our studies revealed potential roles for cytokines/chemokines in HFRS/HPS immunopathogenesis and point to intriguing possibilities for the possible differentiation of infected macrophages to dendritic-like cells.     

Coexistence of fusion receptors for human T-cell leukemia virus type-I (HTLV-I) and human immunodeficiency virus type-1 (HIV-1) on MOLT-4 cells.

Human immunodeficiency virus type-1 (HIV-1) and human T-cell leukemia virus type-I (HTLV-I) have a similar tropism for target cell types, especially for CD4+ T cells. In this study, we provide evidence that receptors of these two viruses exist independently on the target cell. We established an HTLV-I-producing CD8+ T cell line (ILT-8M2) with a remarkable cell fusion capacity. When cocultured with MOLT-4 cells, ILT-8M2 cells induced giant syncytia more efficiently than any other tested HTLV-I-producer cell lines. In contrast to other HTLV-I-producers, ILT-8M2 cells were minimally susceptible to cytopathic effects of HIV-1 due to very low expression of CD4, although they were able to be persistently infected by HIV-1. The indicator MOLT-4 cells are known to respond well to HIV-1-induced cell fusion, but they lose this ability if they become persistently infected with HIV-1 because of the reduction of CD4 receptor expression. ILT-8M2 was, however, still capable of inducing syncytia with the MOLT-4 cells persistently infected by HIV-1 (MOLT-4/IIIB). This syncytium formation was dependent on the HTLV-I-envelope, as it was inhibited by HTLV-I-positive human sera or a monoclonal antibody to HTLV-I gp46 but not by monoclonal antibodies to HIV-1 gp120 or CD4. Moreover, ILT-8M2 cells persistently infected by HIV-1 (ILT-8M2/IIIB) induced both HTLV-I- and HIV-1-mediated syncytia with uninfected MOLT-4 cells. These results suggest that HTLV-I induces cell fusion utilizing receptors on the target cells independent of HIV-1-receptors.     

Distinct Mechanisms Trigger Apoptosis in Human Immunodeficiency Virus Type 1-Infected and in Uninfected Bystander T Lymphocytes

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decrease of CD4⁺ T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or in uninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type 1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we have determined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-V assays in different cell subpopulations, i.e., in CD4⁺ or CD8⁺ T cells that were GFP positive or negative. After HIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly in infected CD4⁺ peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages in the culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis in HIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis results from at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD4⁺ T lymphocytes and (ii) indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.     

Heat Shock Cognate Protein 71-Associated Peptides Function as an Epitope for Toxoplasma gondii-Specific CD4+ CTL

HLA-DR-restricted CD4⁺ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4⁺ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4⁺ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4⁺ CTL.     

Primary HIV-1 infection of human CD4+ T cells passaged into SCID mice leads to selection of chronically infected cells through a massive fas-mediated autocrine suicide of uninfected cells

We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.     

IFN-gamma and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells.

Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.     

Evidence that platelet-derived microvesicles may transfer platelet-specific immunoreactive antigens to the surface of endothelial cells and CD34+ hematopoietic stem/ progenitor cells--implication for the pathogenesis of immune thrombocytopenias

The pathogenesis and tissue damage that accompanies destruction of platelets in immune thrombocytopenias (IT) is still not understood very well and in addition to platelets, other cells (e.g. endothelial cells, CD34+ hematopoietic stem/progenitors) may also become affected. Based on our previous work that platelet antigens (e.g., CD41) may be transferred by platelet-derived microvesicles (PMV) to the surface of other cells, we asked if platelet derived-antigens, especially those that are involved in the formation of anti-platelet antibodies in IT (e.g., against antigen HPA 1 a) could be also transferred by similar mechanism. To address this issue normal human CD34+ cells, human umbilical vein-endothelial cells (HUVEC) and monocytic cell line THP-1 were incubated with PMV derived from HPA1a+ donors. We noticed that the HPA1a antigen is highly expressed on PMV-derived from the HPAla positive platelets and is transferred in PMV-dependent manner to the surface of CD34+ cells, HUVEC and monocytic THP-1 cells. These cells covered with HPA1a positive PMV but not by PMV derived from HPAla negative platelets reacted with anti-HPA1a antibodies derived from the alloimmunized pregnant women. More importantly, human hematopoietic cells that were preincubated with HPA1a+ PMV and subsequently exposed to anti-HPA 1 a serum and human NK cells, become subject to elimination by antibody dependent cell cytotoxicity ADCC. Thus, we postulate that PMV-dependent transfer of antigens may playing an important role in "expanding" the population of target cells that may be affected by anti-platelet antibodies and explain several pathologies that accompany IT (e.g. damage of endothelium, cytopenias).     

Influence of products of bacterial origin on the expression of surface molecules in monocyte-derived and endothelial cells

AIM: To study the influence of lypopolysaccharide (LPS) of Gram-negative bacterium (Escherichia coli O55:B5) and lysate of Gram-positive bacteria (Streptococcus pyogenes - group A, type M1, strain 40/58) on the level of expression of important surface molecules of monocyte-derived cells from continuous cell line THP-1 and endothelial cells from continuous cell line EA.hy 926. MATERIALS AND METHODS: Expression of surface molecules HLA-DR, CD11b, CD14, CD16, CD32, and CD54 was assessed using FITC- or PE-labeled monoclonal antibodies (Beckman Coulter, USA). Intensity of fluorescence was measured by flow cytometer Epics Altra manufactured by Beckman Coulter (USA). RESULTS: Studied components of Gram-positive and Gram-negative bacteria stimulated expression of CD14, CD16, CD32, and CD54 molecules on cells from THP-1 line; incubation of cells from EA.hy 926 line in the presence of the same bacterial components increased expression levels of CD54 and HLA-DR molecules. CONCLUSION: Endothelial cells of EA.hy 926 line was less sensitive to LPS of E. coli and lysate of S. pyogenes compared to monocyte-derived cells of THP-1 line. Usage of THP-1 cells allowed to reveal differences between effects of components of Gram-positive and Gram-negative bacteria. The stimulating effect of LPS was more pronounced compared to effect of S. pyogenes lysate in relation to expression of HLA-DR, CD11b, and CD54 molecules, whereas lysate of S. pyogenes better stimulated expression of CD14, CD16, and CD32 molecules.