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.

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.

AP-1 and Cbfa/runt physically interact and regulate parathyroid hormone-dependent MMP13 expression in osteoblasts through a new osteoblast-specific element 2/AP-1 composite element.

The expression of MMP13 (collagenase-3), a member of the matrix metalloproteinase family, is increased in vivo as well as in cultured osteosarcoma cell lines by parathyroid hormone (PTH), a major regulator of calcium homeostasis. Binding sites for AP-1 and Cbfa/Runt transcription factors in close proximity have been identified as cis-acting elements in the murine and rat mmp13 promoter required for PTH-induced expression. The cooperative function of these factors in response to PTH in osteoblastic cells suggests a direct interaction between AP-1 and Cbfa/Runt transcription factors. Here, we demonstrate interaction between c-Jun and c-Fos with Cbfa/Runt proteins. This interaction depends on the leucine zipper of c-Jun or c-Fos and the Runt domain of Cbfa/Runt proteins, respectively. Moreover, c-Fos interacts with the C-terminal part of Cbfa1 and Cbfa2, sharing a conserved transcriptional repression domain. In addition to the distal osteoblast-specific element 2 (OSE2) element in the murine and rat mmp13 promoter, we identified a new proximal OSE2 site overlapping with the TRE motif. Both interaction of Cbfa/Runt proteins with AP-1 and the presence of a functional proximal OSE2 site are required for enhanced transcriptional activity of the mmp13 promoter in transient transfected fibroblasts and in PTH-treated osteosarcoma cells.

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.

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.

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.

Definition of peptide binding motifs amongst the HLA-A*30 allelic group.

HLA class I molecules present endogenously processed peptide ligands for surveillance by the T-cell receptor. This potentially immunogenic surface of HLA and peptide is a consequence of the polymorphism found within the HLA molecule and its preference for ligand binding together with peptide conformation within the binding groove. To investigate the relation between the polymorphic differences between some closely related HLA alleles and their effect on peptide preference, transfectants were established, each containing one of four allelic variants of HLA-A*30. Peptides from all four transfectants were eluted, and both individual ligands and peptide pools were sequenced. The data shows two distinct peptide motifs which distinguish A*3001 from the other three known A*30 variants. Differences in preferences at minor positions within the peptide sequence were noted between A*3002, A*3003 and A*3004, providing additional evidence of the implications of sequence polymorphism to HLA function.

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.

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.

TGF-beta-independent induction of immunogenicity by decorin gene transfer in human malignant glioma cells.

Ectopic expression of the proteoglycan, decorin, abrogates the growth of experimental C6 gliomas in the rat. Since gliomas release large amounts of transforming growth factor-beta (TGF-beta) and since decorin is a TGF-beta antagonist, decorin gene transfer-mediated abrogation of glioma growth in vivo may involve enhanced immunogenicity of the tumor cells. Here, we report that human glioma cells stimulate alloreactive immune responses when engineered to express decorin whereas parental glioma cells are non-immunogenic in vitro. The alloreactive immune response is mediated by CD8+ and CD4+ T cells as well as by NK cells. The immunosuppression exerted by parental or mock-transfected glioma cells is mediated by soluble factors and can in part be mimicked by exogenous TGF-beta. However, neutralizing anti-TGF-beta antibodies do not reverse glioma-mediated immunosuppression, suggesting that decorin abrogates glioma-induced immune cell inhibition by interfering with the activity of other, so far unidentified glioma-secreted mediators. We conclude that enhanced immunogenicity may mediate the antineoplastic effects of decorin gene therapy for malignant glioma but that factors other than TGF-beta may be responsible for glioma-induced immunosuppression.

The role of peptide presentation in the physiological function of HLA-G.

The HLA-G gene gives rise to six differently spliced mRNAs. The membrane bound HLA-G1 molecule containing all three extracellular domains presents peptides that follow motif requirements similar to those of classical HLA class I molecules. This isoform is also capable of inhibiting Natural Killer (NK) cells, but is only efficiently transported to the cell surface when peptides are provided in the endoplasmic reticulum. In the absence of sufficient peptide supply to the ER a small molecule of 18-kDa is transported to the cell surface in HLA-G transfectants of LCL721.221 cells. HLA-G transfectants with impaired ER peptide supply are nevertheless protected from NK lysis.

Alloreactivity as a source of high avidity peptide-specific human CTL.

PBL from HLA-A2- or HLA-A3- donors were stimulated with synthetic peptide libraries fitting HLA-A2 or HLA-A3 motifs and presented on HLA-A2- or HLA-A3-expressing TAP- cells. Peptide library-specific allorestricted CTL were found to constitute up to half the alloreactive CTL response and occurred at twofold lower frequency than autologous peptide library-specific CTL. This indicates that positive selection by one particular MHC class I molecule is not absolutely essential for the generation of CTL restricted to the same molecule. However, positive selection increases their frequency. The CTL obtained differed greatly both with respect to peptide dependency and peptide specificity. Determination of the peptide avidity for one representative CTL clone, 10F4, proved that the method described here allows the stimulation of high avidity cytotoxic T cells. This approach involving in vitro stimulation of T cells restricted toward a MHC molecule that was not present during their negative selection might therefore offer the possibility of isolating CTL against self and foreign peptides with varying avidities. Such T cells might indeed be useful for tumor immunotherapy.

Allo- and self-restricted cytotoxic T lymphocytes against a peptide library: evidence for a functionally diverse allorestricted T cell repertoire.

BALB/c-derived spleen cells were depleted of cytotoxic T lymphocytes (CTL) recognizing allogeneic (H2b) and TAP-negative cells followed by stimulation with the same cells loaded with a synthetic library binding to H2-Kb. The resulting CTL lines were found to differ widely in peptide specificity and to exhibit an avidity towards the library as that demonstrated for syngeneic CTL. These results demonstrate that positive selection in the context of a certain MHC molecule does not seem to be required for generating high-avidity TCR that are restricted by the same molecule. However, positive selection increases the frequency of such CTL. By raising T cell lines from a repertoire which did not undergo negative selection by the restriction element in question, it becomes possible to produce effective self-peptide/ MHC as well as nonself-peptide/MHC-specific CTL as tools for adoptive tumor immunotherapy.

[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.

Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in Western patients with cirrhosis.

The aim of this study was to identify high-risk patients for hepatocellular carcinoma (HCC). Among 151 patients with histologically proven cirrhosis hospitalized from 1987 to 1990 and prospectively followed-up until June 1994, 31 developed HCC. We assessed the predictive value of 22 variables recorded at enrollment for HCC occurrence by the log rank test and the Cox proportional hazards model. Six clinical and biological variables summarized predictive information of HCC: age > or = 50 years (P = .01), male (P = .01), large esophageal varices (EV) (P = .03), prothrombin activity < 70% (P = .04), serum alpha-fetoprotein (AFP) > or = 15 ng/L (P = .06), and anti-hepatitis C virus antibodies (P = .08). A clinicobiological predictive score identified two groups of patients at low (n = 67; 3-year cumulative incidence, 0%) and high risk for HCC (n = 84; 3-year cumulative incidence, 24%). The predictive value of this score was confirmed using an independent population of 49 patients with cirrhosis. Furthermore, liver large-cell dysplasia (LCD) had an additional predictive value in high-risk patients (P = 10(-4), which thus helped to define a subgroup at very high risk for HCC (n = 12; 3-year cumulative incidence, 72%). In Western patients with cirrhosis, a limited number of usual variables can identify a group of patients at high risk for HCC. Among these patients, liver biopsy allows for the determination a subgroup of patients at very high risk for HCC requiring intensive screening or preventive measures.

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.

Serum bile acids and cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological features.

Cholestasis is a biochemical and/or histological feature observed in some patients with alcoholic liver disease and is mainly related to alcoholic hepatitis. Accumulation of bile acids in the liver could be pathogenic in alcoholic hepatitis. The aim of this study was to assay serum bile acids in patients with alcoholic hepatitis and to assess the relationship between these parameters, the usual liver tests and the histological features of alcoholic hepatitis. Thirty-six patients (median 51 years, 19 females and 17 males) with biopsy-proven alcoholic hepatitis were included in the study. Cirrhosis was present in 27 patients. Serum bile acids were assayed by high performance liquid chromatography. Three histological scores (alcoholic hepatitis, fibrosis, and cholestasis) were established on each liver sample by two independent pathologists. Serum bile acid concentrations were increased in 35 patients (97%). The median concentration of total serum bile acids was 41.6 mumol/l (range 3-293), with an increase in primary bile acids (95.7% of total bile acids), mainly chenodeoxycholic acid (median 27.5 mumol/l, range 3-184). In contrast, serum bilirubin levels were increased in only 26 patients (72%). Histological cholestasis was present in 14 patients (38%). There was no significant correlation between the alcoholic hepatitis and cholestasis scores (r = 0.01, p = 0.9). A significant correlation was noted between the alcoholic hepatitis score and serum total bile acid (r = 0.34, p = 0.04), cholic acid (r = 0.38, p = 0.03) and chenodeoxycholic acid (r = 0.32, p = 0.05) levels.(ABSTRACT TRUNCATED AT 250 WORDS)