Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells.

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.

Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.

Immunostimulatory properties of dendritic cells (DCs) are linked to their maturation state. Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans. Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects. In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells. When pre- and postimmunization T cells were boosted in culture, there were greater numbers of MP-specific major histocompatibility complex tetramer-binding cells after immunization, but these had reduced interferon production and lacked killer activity. These data demonstrate the feasibility of antigen-specific inhibition of effector T cell function in vivo in humans and urge caution with the use of immature DCs when trying to enhance tumor or microbial immunity.

Human histocompatibility leukocyte antigen (HLA)-G molecules inhibit NKAT3 expressing natural killer cells.

The crucial immunological function of the classical human major histocompatibility complex (MHC) class I molecules, human histocompatibility leukocyte antigen (HLA)-A, -B, and -C, is the presentation of peptides to T cells. A secondary function is the inhibition of natural killer (NK) cells, mediated by binding of class I molecules to NK receptors. In contrast, the function of the nonclassical human MHC class I molecules, HLA-E, -F, and -G, is still a mystery. The specific expression of HLA-G in placental trophoblast suggests an important role for this molecule in the immunological interaction between mother and child. The fetus, semiallograft by its genotype, escapes maternal allorecognition by downregulation of HLA-A and HLA-B molecules at this interface. It has been suggested that the maternal NK recognition of this downregulation is balanced by the expression of HLA-G, thus preventing damage to the placenta. Here, we describe the partial inhibition of NK lysis of the MHC class I negative cell line LCL721.221 upon HLA-G transfection. We present three NK lines that are inhibited via the interaction of their NKAT3 receptor with HLA-G and with HLA-Bw4 molecules. Inhibition can be blocked by the anti-NKAT3 antibody 5.133. In conclusion, NK inhibition by HLA-G via NKAT3 may contribute to the survival of the fetal semiallograft in the mother during pregnancy.

Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I-restricted re-presentation of gp96-associated peptides and CTL activation; non-receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1.

The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8(+) cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4(+) T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4(+) T cells, EBNA1 is preferentially recognized. We present evidence that the CD4(+) response may provide a protective role, including interferon gamma secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II-mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4(+) T cell lines to recognize autologous B-LCL-expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4(+) T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4(+) T cell immunity be enhanced to prevent and treat EBV-associated malignancies.

Antiviral immune response in patients with multiple sclerosis and healthy siblings.

The objective of this study was to determine the immune responses to candidate viral triggers of multiple sclerosis in patients and healthy siblings raised in the same family household. Virus antigen-specific IgG responses to Epstein-Barr virus-derived gene products as well as to human herpersvirus-6, human cytomegalovirus, and measles virus were evaluated in 25 multiple sclerosis patients and compared with 49 healthy full-siblings. IgG responses to the latent Epstein-Barr virus-encoded nuclear antigen-1 (EBNA1) were selectively increased in individuals with multiple sclerosis compared with their unaffected siblings. We conclude that elevated IgG responses towards EBNA1 are associated with the development of multiple sclerosis.

Nonclassical HLA-G molecules are classical peptide presenters.

BACKGROUND: The physiological functions of the classical HLA (human leukocyte antigen) molecules, HLA-A, HLA-B and HLA-C, are to present peptides to T cells and to inhibit the activity of natural killer cells. In contrast, the functions of nonclassical HLA-molecules, such as HLA-E, HLA-F and HLA-G, remain to be established. The expression of HLA-G is largely limited to the placental trophoblast, where it might mediate protection of the fetus from rejection by the mother. Achieving the aim of understanding the function of HLA-G should be facilitated by information on the biochemical properties of HLA-G molecules, especially on their potential ability to act as peptide receptors. RESULTS: To study peptide presentation by HLA-G, we used stably transfected LCL721.221 cells as a source of HLA-G molecules and analysed the spectrum of extracted peptides by individual and pool sequencing. Our results indicate that HLA-G molecules, like classical HLA molecules, are associated with a wide array of peptides derived from cellular proteins. Peptides presented by HLA-G usually consisted of 9 amino acids, and adhered to a specific sequence motif, with anchor residues at position 2 (isoleucine or leucine), position 3 (proline) and the carboxy-terminal position 9 (leucine). Thus, the HLA-G peptide ligand motif follows the principles of classical HLA motifs, although it displays its own unique features. Peptide-binding assays indicated that two of the three anchor residues were sufficient for binding, and that the three natural HLA-G ligands that we identified bound, not only to HLA-G, but also to HLA-A2. This was not surprising, because the binding pockets of HLA-A2 and HLA-G overlap in their ability to recognize anchor residues at positions 2 and 9. Likewise, some, but not all, HLA-A2 peptide ligands could also bind to HLA-G. CONCLUSIONS: Nonclassical HLA-G molecules present peptides essentially in the same way as classical HLA molecules do. We determined the peptide motif that is specifically recognized by HLA-G; its basic features are described by the sequence XI/LPXXXXXL: This information should help to elucidate the physiological role of HLA-G molecules at the fetal-maternal interface. Most likely, this role is to protect fetal cells from lysis by natural killer cells, and possibly to present foreign peptides to a class of T cells that has not yet been identified.

EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) maintains the viral episome in all host cells infected with EBV. Recently, EBNA1 was found to be the main EBV latency antigen for CD4+ T cells and could be recognized in cultures from all donors tested. We now identify a polarized Th1 phenotype and obtain evidence for its presence in vivo. When T cells were stimulated with dendritic cells infected with vaccinia vectors expressing EBNA1, 18 of 19 donors secreted IFN-gamma, whereas only two of 19 secreted IL-4. Magnetic selection was then used to isolate cells from fresh blood based on EBNA1-induced cytokine production. Specific IFN-gamma CD4+ cell lines were established from six of six donors and IL-4 lines from three of six. Only the Th1 lines specifically lysed targets expressing three different sources of EBNA1 protein. When the IgG isotype of EBNA1 plasma Ab's was tested, most specific Ab's were IgG1 and of a high titer, confirming a Th1 response to EBNA1 in vivo. Ab's to other microbial antigens generally were not skewed toward IgG1. Given emerging evidence that Th1 CD4+ T cells have several critical roles in host defense to viral infection and tumors, we propose that EBNA1-specific CD4+ Th1 cells contribute to resistance to EBV and EBV-associated malignancies.

Immune surveillance of intracellular pathogens via autophagy.

MHC class II molecules are thought to present peptides derived from extracellular proteins to CD4+ T cells, which are important mediators of adaptive immunity to infections. In contrast, autophagy delivers constitutively cytosolic material for lysosomal degradation and has so far been recognized as an efficient mechanism of innate immunity against bacteria and viruses. Recent studies, however, link these two pathways and suggest that intracellular cytosolic and nuclear antigens are processed for MHC class II presentation after autophagy.

Autophagy and Mammalian Viruses: Roles in Immune Response, Viral Replication, and Beyond.

Autophagy is an important cellular catabolic process conserved from yeast to man. Double-membrane vesicles deliver their cargo to the lysosome for degradation. Hence, autophagy is one of the key mechanisms mammalian cells deploy to rid themselves of intracellular pathogens including viruses. However, autophagy serves many more functions during viral infection. First, it regulates the immune response through selective degradation of immune components, thus preventing possibly harmful overactivation and inflammation. Additionally, it delivers virus-derived antigens to antigen-loading compartments for presentation to T lymphocytes. Second, it might take an active part in the viral life cycle by, eg, facilitating its release from cells. Lastly, in the constant arms race between host and virus, autophagy is often hijacked by viruses and manipulated to their own advantage. In this review, we will highlight key steps during viral infection in which autophagy plays a role. We have selected some exemplary viruses and will describe the molecular mechanisms behind their intricate relationship with the autophagic machinery, a result of host-pathogen coevolution.

Contrasting roles of dendritic cells and B cells in the immune control of Epstein-Barr virus.

The human gamma-herpesvirus, Epstein-Barr virus (EBV), has growth-transforming potential in vivo and in vitro. Despite this, most healthy carriers remain free of EBV-associated malignancies because of effective T cell-mediated immune control of the virus. A better understanding of these highly efficient control mechanisms is important in the development of new treatment strategies for EBV-associated malignancies. A rational approach to EBV immunotherapy requires answering two questions about the initiation of the protective EBV-specific immune response. The first question is, what is the antigen-presenting cell responsible for priming EBV specific immunity? Second, which viral antigen is central to protective EBV adaptive immunity seen in healthy carriers of the virus? We provide evidence in this review that dendritic cells rather than EBV-transformed B cells are responsible for orchestrating protective EBV immunity and that the EBV nuclear antigen 1 (EBNA1)-specific CD4+ T cell response probably plays a role in resistance against all types of EBV-associated malignancies in healthy carriers. This implies that EBNA1 targeting to dendritic cells should be a component of vaccine and immunotherapy development against EBV-associated malignancies.

Generation of high quantities of viral and tumor-specific human CD4+ and CD8+ T-cell clones using peptide pulsed mature dendritic cells.

CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-gamma intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy.

Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120.

HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120.

Peptide presentation and NK inhibition by HLA-G.

The expression of the nonclassical MHC class Ib molecule HLA-G is nearly exclusively restricted to the feto-maternal interface during pregnancy. There it probably serves the same physiological functions already known for classical MHC class I molecules; these include peptide presentation, natural killer cell (NK) inhibition and probably also T cell restriction. In this study a comparison between HLA-G and HLA-A2 as far as the amount and complexity of bound peptides is concerned revealed no significant differences. The peptide motif of HLA-G, as determined by analysis of naturally eluted peptides allows the construction of a peptide library that is efficient in binding to HLA-G and thereby confirms the rules of peptide binding to this nonclassical MHC class I molecule. In addition, we demonstrate that the inhibition of NK cells by HLA-G varies remarkably among the NK repertoires of different donors. The function of HLA-G as a survival factor in the development of the fetus during pregnancy is discussed in detail.

[Severe hepatic steatosis: a cause of sudden death in the alcoholic patient]. / La stéatose hépatique massive: une cause de décès brutal chez le malade alcoolique.

The authors report the cases of 4 patients with heavy chronic alcoholic intake who presented with hepatomegaly and jaundice without obvious hepatic failure and who died rapidly. In all 4 cases, histological examination of the liver showed massive microvesicular and macrovesicular steatosis involving approximately 100% of hepatocytes and, in 2 cases, minimal lesions of alcoholic hepatitis. Histochemical study, performed in 3 cases, showed that steatosis was constituted of triglycerides only, and that hepatic glycogen was completely depleted in 2 of 3 cases. No obvious cause of death was found in these 4 patients. Shortly, before their death, the 4 patients had increased their ethanol and decreased their food intake. The authors suggest that death as well as microvesicular steatosis could have be due to acute mitochondrial dysfunction.

[Role of hepatitis C virus in the genesis of hepatic lesions observed in alcoholic patients with liver cirrhosis]. / Rôle du virus de l'hépatite C dans la genèse des lésions hépatiques observées chez des malades alcooliques atteints de cirrhose.

The aim of study was to assess the role of hepatitis C virus (HCV) infection in 164 alcoholic cirrhotic patients. We studied the prevalence of anti-HCV antibodies using ELISA and RIBA first and second generation tests. Twenty-two % of the patients had anti-HCV antibodies detected by ELISA 2, RIBA 2 test was positive in 10% of the patients and indeterminate in 3%. We compared epidemiological, biological and histological characteristics according to the results of the tests. By comparing ELISA 2-RIBA 2 positive patients to ELISA 2 negative patients, we observed, in the former, a) a higher serum aminotransferase activity, b) a lower serum gammaglutamyl transpeptidase activity, and c) a lower histological score of alcoholic hepatitis. In addition, in a group of ELISA 2 positive RIBA 2 negative patients, the values were intermediate between those of the two former groups. However, most of these patients had a negative third generation ELISA test. The whole results suggest that HCV is likely to play a role in the pathogenesis of liver damage in a high number of alcoholic cirrhotic patients.

[Comparison of six potential serum markers of hepatic fibrosis in alcoholic disease of the liver]. / Comparaison de 6 marqueurs sériques potentiels de la fibrose hépatique au cours de la maladie alcoolique du foie.

The aim of this study was to compare 6 potential serum markers for hepatic fibrosis in patients with alcoholic liver disease. Ninety-three patients (50 +/- 11 years old, 62 males) with biopsy-proven alcoholic liver disease were included in the study. A liver biopsy and serum assays of type I, type III and type IV collagens, N-terminal peptide of type III procollagen, laminin (by radioimmunoassays) and apolipoprotein A1 (by nephelometry) were performed in all patients. A histological score of hepatic fibrosis was established. Alcoholic hepatitis lesions and perisinusoidal fibrosis were assessed separately. A significant correlation was found between the score of hepatic fibrosis and serum levels of type I collagen (r = 0.44, P < 10(-3)), type III collagen (r = 0.36, P < 10(-2)), N-terminal peptide of type III procollagen (r = 0.50, P < 10(-3)), type IV collagen (r = 0.44, P < 10(-3)), laminin (r = 0.50, P < 10(-3)), and apolipoprotein A1 (r = 0.21, P < 0.05). After adjustment for the presence of lesions of alcoholic hepatitis and perisinusoidal fibrosis (partial correlation), serum levels of type I collagen, type III collagen, N-terminal peptide of type III procollagen, type IV collagen, and laminin remained significantly correlated with the score of hepatic fibrosis; in contrast, correlation with serum apolipoprotein A1 was no longer significant. Serum levels of N-terminal peptide of type III procollagen, type IV collagen and laminin were significantly higher in patients with perisinusoidal fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Struma ovarii or malignant ovarian goiter. A case]. / Les struma ovarii ou goitres ovariens malins. Une observation.

BACKGROUND: We report a case of follicular struma ovarii observed in an ovary teratoma without metastatic dissemination. CASE REPORT: A right ovarian tumor was discovered at ultrasound examination in a 31-year-old woman complaining of low abdominal pain. The patient underwent laparoscopic exploration and a 4-cm cystic mass of the right ovary was removed. Microscopic examination showed a malignant struma ovarii of the follicular type with vascular space invasion; other teratomous elements were identified. Immunohistochemical staining for thyroglobulin confirmed the nature of the tumor. The patient was treated by complete right ovariectomy followed by total thyroidectomy and administration of radioactive iodine (99 mCi I-131). Repeat I-131 body scan performed at 6 months was normal. DISCUSSION: Struma ovarii is a rare type of ovarian teratoma, consisting mainly of thyroid tissue. The incidence of malignant struma ovarii is below 1% and fewer than two dozen cases with distant metastases have been reported. The major problem associated with struma ovarii has been the establishment of criteria for malignancy.

Dysregulated Epstein-Barr virus infection in patients with CIDP.

Ubiquitous viruses have frequently been proposed as a cause or trigger of chronic immune-mediated diseases. Infections are reported to be temporally associated with clinical exacerbations in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). We examined immunological parameters of herpesvirus infections in untreated patients with CIDP compared to demographically matched controls. Patients with CIDP were uniformly seropositive for EBV-specific IgG and the disease was associated with a moderately enhanced IgG reactivity to EBV-encoded antigens expressed during both B cell transformation and productive viral replication. Moreover, cellular EBV copy numbers were 3-fold increased in patients with CIDP. In contrast, humoral immune responses to other herpesviruses (HCMV, HSV) as well as virus-specific IgM responses were unchanged in CIDP. These data indicate that host-pathogen interactions during chronic EBV infection are dysregulated in treatment-naïve patients with CIDP.