Cyclosporin A promotes spontaneous outgrowth in vitro of Epstein-Barr virus-induced B-cell lines

Cyclosporin A (CSA) is a fungal metabolite which exerts profound effects on the immune system and has potential as a selective immuno-suppressive agent. Clinical trials with human renal allograft recipients have confirmed this potential but there have been disturbing reports of lymphoma in a significant number of patients. Despite extensive animal studies, the specificity of this drug for human lymphocyte subpopulations is largely unknown. We demonstrate here that in vitro CSA has no apparent effect on Epstein-Barr virus (EBV)-induced B-lymphocyte activation, but totally inhibits the T-cell dependent pokeweed mitogen (PWM) B-cell response. In addition, CSA markedly facilitates the outgrowth of B-lymphoblastoid cell lines from both EBV-infected and non-infected lymphocytes of EBV immune donors cultured in vitro. These results indicate that CSA can interfere with the lymphocytes normally responsible for maintaining the life-long carrier state initiated by primary infection with EBV, allowing outgrowth of the persistently infected cells circulating in the peripheral blood.     

Cell lines producing human T-cell lymphoma virus show altered HLA expression

Human T-cell leukaemia/lymphoma virus (HTLV) can be identified in fresh and cultured T-lymphocytes from patients with adult T-cell malignancies. HLA typing of the peripheral blood lymphocytes and cultured cell lines from the patient from which the virus was originally isolated suggested the expression of additional HLA-A and -B locus antigens on the HTLV positive cultured T-cells that were not present on the EBV transformed B-cell line or on the peripheral blood lymphocytes. Peripheral blood lymphocytes (PBLs) and T-cell lines established from patients and cord blood lymphocytes, infected with virus by co-culture with T-cell lines, were typed for HLA antigens with alloantisera and in addition tested for reactivity with a monoclonal antibody (4D12) which recognizes a polymorphic HLA class-I antigen. In all HTLV positive cells, with demonstrable provirus replication, altered HLA alloantigen expression was observed. This may be explained by the observations reported in the accompanying paper which shows homology between the envelope gene region of HTLV and the region of an HLA-B locus gene which codes for the extracellular portion of a class I histocompatibility antigen.     

T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria

Endemic Burkitt's lymphoma, a tumour of children in which B lymphocytes are infected with Epstein-Barr virus (EBV), is common in areas of Africa where malaria is holoendemic. The tumour is characterized by chromosome translocations; usually the terminal portion of chromosome 8 containing the c-myc gene is translocated to chromosome 14, near the enhancer of the immunoglobulin heavy-chain locus. Less frequent are translocations of chromosome 8 to the kappa light-chain locus of chromosome 2 or to the lambda light-chain locus of chromosome 22. In vitro, EBV induces B cells to proliferate and secrete immunoglobulin and antibody. However, in vivo the infected B lymphocytes are under immunological control, so that abnormal proliferation is found only in immunosuppressed patients. Such patients are subsequently liable to develop lymphomas. Burkitt believed that the tumour he had described resulted from interaction between a virus(es) and a "reticuloendothelial system altered by chronic and heavy infection by malarial or other parasites". We report here that during an attack of Plasmodium falciparum malaria, T-cell subpopulations are radically altered so that, in vitro, B lymphocytes infected with EBV proliferate abnormally to secrete large amounts of immunoglobulin and antibody. This phenomenon offers some explanation for the increased incidence of Burkitt's tumour and the high levels of immunoglobulin found in people living in areas where P. falciparum malaria is common.     

HLA-restricted T-cell recognition of Epstein-Barr virus-infected B cells

In mice the cytotoxic T-cell response to several types of virus is influenced by genes within the major histocompatibility complex; in particular, genetic control is exercised at the effector cell level through a requirement that virus-specific cytotoxic T cells recognise viral antigens in association with H-2K and H=2D region gene products on the surface of infected cells. In man the restriction which the analogous HLA-A, -B and -C-region gene products might place on virus-specific T-cell function is still in dispute. The earliest and most controversial evidence concerns the Epstein-Barr virus (EBV), a B lymphotropic agent which causes infectious mononucleosis (IM) and which induces an unusually vigorous T-cell response; cytotoxic T cells from IM patients' blood were shown to be EBV-specific yet, in contrast to mouse systems, apparently free of any obvious HLA restriction. Since then T-cell recognition of EBV-infected B cells has assumed particular significance as a model system for the study of cytotoxic T-cell function in man. This report describes the results of a new approach clearly indicating that HLA-A and -B region products do indeed have a role in this system.     

Replication of Epstein-Barr virus in human epithelial cells infected in vitro

Epstein-Barr virus (EBV), a member of the herpes group of viruses and the aetiological agent of infectious mononucleosis, is usually thought of as a lymphotrophic virus with the ability to transform B lymphocytes. So the association of EBV with nasopharyngeal carcinoma is puzzling, especially given the lack of success of attempts to infect epithelial cells with EBV in culture and the apparent lack of EBV receptors on epithelial cells. Circumvention of the apparent requirement for membrane receptors by techniques of transfection, microinjection and receptor transplantation has clearly demonstrated that there is no inherent barrier to EBV replication in nonlymphoid cells, including epithelial cell types. Our ability routinely to detect EBV DNA by in situ hybridization in epithelial cells of the oropharynx from persons with acute infectious mononucleosis suggests that, in vivo, EBV regularly gains access to and replicates lytically in epithelial cells. We report here in vitro evidence for direct infection by EBV and replication of the virus in cultured normal human epithelial cells.     

Cytotoxic T-cell clones discriminate between A- and B-type Epstein-Barr virus transformants

Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis and is associated with Burkitt's lymphoma and nasopharyngeal carcinoma. The virus is harboured for life in all previously infected individuals and is apparently controlled by a population of EBV-specific memory T lymphocytes, specifically activated to recognize the functionally defined lymphocyte-detected membrane antigen. Two types (A and B) of EBV have been identified that show DNA sequence divergence within the BamH1 WYH region of the genome encoding the transformation-associated antigen, Epstein-Barr nuclear antigen 2 (EBNA 2) (ref. 4). To define the function of EBNA 2 in T-cell recognition, we have compared the ability of EBV-specific cytotoxic T-cell clones to distinguish between autologous B lymphocytes transformed by A- or B-type virus. We have now isolated both CD4 and CD8 cytotoxic T-cell clones that recognize autologous A-type but not B-type transformed lymphoblastoid cell lines, thus providing the first evidence that EBV-specific T-cell recognition can be mediated by EBNA 2. As this antigen is not expressed in Burkitt's lymphoma, this finding explains the failure of EBV-specific T-cell surveillance to eliminate the tumour.     

High tyrosine kinase activity in normal nonproliferating cells

Protein phosphorylation at serine and threonine residues has been implicated in the regulation of many cellular processes. More recently, tyrosine residue phosphorylation has been shown to be associated with stimulation of cell proliferation, including viral transformation and stimulation by epidermal growth factors (EGF), platelet-derived growth factor (PDGF) and other compounds related to cellular growth such as insulin and dimethyl sulphoxide. To compare protein kinases and phosphoproteins of normal and leukaemic human haematopoietic cells in vivo and in vitro, we first have investigated the percentages of phosphoserine, phosphothreonine and phosphotyrosine obtained after hydrolysis of proteins from different blood cell fractions phosphorylated in vitro. We report here that phosphotyrosine formed less than 1% of the soluble fractions from polymorphonuclear cells, mononuclear cells (80% circulating lymphocytes, 20% monocytes), blood platelets and red blood cells (not shown). Surprisingly, high percentages of phosphorylated tyrosine were found only in the particulate fractions from non-proliferating anuclear cells, platelets and red blood cells.     

Corticotropin releasing factor induction of leukocyte-derived immunoreactive ACTH and endorphins

Human peripheral leukocytes infected by virus or treated with endotoxin will, like unstimulated mouse spleen macrophages, synthesize immunoreactive corticotrophin (ir-ACTH) and endorphins. The ir-ACTH produced appears to be identical with authentic ACTH, while enough of the material has been produced in hypophysectomized mice infected with virus to demonstrate a steroidogenic response. Because the production of ACTH by in vivo pituitary cells and by leukocytes is suppressed by dexamethasone both in vitro and in vitro, suggesting that the production of ACTH and endorphins by leukocytes is indeed controlled, we have investigated the effects of corticotropin releasing-factor (CRF), which is known to regulate the pituitary production of both ACTH and beta-endorphin. We now report that the production of ACTH and endorphins by leukocytes is indeed induced by synthetic CRF and, in turn, suppressed by dexamethasone, suggesting that, as in pituitary cells, the proopiomelanocortin (POMC) gene may be expressed and similarly controlled in leukocytes.     

Isolation of HTLV-transformed B-lymphocyte clone from a patient with HTLV-associated adult T-cell leukaemia

The human T-cell leukaemia/lymphoma virus (HTLV) is an exogenous retrovirus which has been associated with adult T-cell leukaemia/lymphoma (ATL). This malignancy of T lymphocytes is endemic to southern Japan, the West Indies, and to a lesser extent, the Middle East, Central Africa and the southeastern United States. ATL cells from patients of diverse geographical origins have been found to be infected with HTLV-1 (ref.6). HTLV is normally tropic for mature T lymphocytes, especially those expressing the helper-inducer surface antigen phenotype (OKT4 or Leu-3-positive), and the neoplastic T cells infected with HTLV generally express receptors for T-cell growth factor (detected by reactivity with anti-Tac antibody). However, we report here the isolation of a HTLV-infected B-lymphocyte clone from the peripheral blood of a patient with ATL. This clone is cytogenetically normal and is not infected with Epstein-Barr virus (EBV). Co-culture of cells from this clone with cord blood lymphocytes resulted in transmission of HTLV and the immortalization of either T or B lymphocytes. These results suggest that HTLV may be associated with a broader range of host cells than previously recognized.     

Epstein-Barr virus genome-positive T lymphocytes in a boy with chronic active EBV infection associated with Kawasaki-like disease

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus and an aetiological agent of infectious mononucleosis, has a unique tropism for B lymphocytes. Clinical and laboratory features of chronic active EBV infections are chronic or persistent infectious mononucleosis-like symptoms and high antibody titre against early antigens (EA). Kawasaki disease (KD), aetiology unknown, is thought to be self-limited immunologically mediated vasculitis. Clinical features of KD are fever, rash, mucositis, lymphadenopathy and coronary artery damage. We report here a child with chronic active EBV infection accompanied by dilatation of coronary arteries. All the EBV-determined nuclear antigen (EBNA)-positive lymphocytes had exclusively CD4 antigen, as revealed by dual staining immunofluorescence analysis. Southern blot hybridization showed that the purified CD4+ cells harboured EBV genome.     

Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.     

Epstein-Barr virus transforms precursor B cells even before immunoglobulin gene rearrangements

The very early stages of the human B-cell differentiation pathway are poorly understood, primarily because of the lack of appropriate permanent cell lines. Epstein-Barr virus (EBV) is a putative human oncogenic virus which transforms human B cells in vitro into continuously proliferating cells. It has been believed that EBV transforms mature B cells, but recently, transformation of immature pre-B-cell lines has been reported, suggesting that EBV might also transform cells much earlier in the B-cell lineage. We report here the establishment of cell lines transformed by EBV at various stages of the B-cell differentiation pathway. Interestingly, two lines showed the complete absence of immunoglobulin synthesis and the lack of immunoglobulin gene rearrangement despite containing EBV genome and surface markers of B cells. Our results indicate that EBV can infect and transform cells of the B lymphocyte lineage even before immunoglobulin gene rearrangement.     

Unique cell lines harbouring both Epstein-Barr virus and adult T-cell leukaemia virus, established from leukaemia patients

Members of three distinct classes of animal virus have been associated with naturally occurring neoplasias in man: Epstein--Barr virus (EBV), a DNA virus belonging to herpesvirus group, papillomavirus, and a novel human RNA (retro) virus, human T-cell leukemia virus or adult T-cell leukaemia (ATL) virus. We have established seven continuous cell lines from ATL patients and 0.1-7% of these cells consistently express ATL-specific antigens (ATLA). EBV-associated nuclear antigen (EBNA) is also found in more than 90% of these cells. We have cloned cells from two of these lines and show here that both ATLA and EBNA were present in the same B-cell clone carrying surface immunoglobulin (sIg).     

Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes

Epstein-Barr virus (EBV), a herpes virus, infects human B lymphocytes in vitro and efficiently immortalizes them. About 10 of the approximately 100 genes of EBV are expressed in recently immortalized B cells and although there is circumstantial evidence that at least three of these may contribute to the process of immortalization, there is no direct evidence that any particular gene is required. We have developed a genetic analysis of EBV that uses a transformation-defective strain of the virus as a helper virus in conjunction with DNA that contains all of the viral cis-acting elements required for replication, cleavage and packaging during the lytic phase of the viral life cycle. This DNA can include viral genes required for immortalization that complement the transformation-defective virus strain. The DNA can be amplified and packaged by the products of the helper virus and the packaged DNA is infectious. We have analysed two viral genes expressed in immortalized cells and find that the gene encoding EBV nuclear antigen-2 is required for immortalization, whereas the gene for the EBV nuclear antigen leader protein is not.     

Effect of recombinant soluble CD4 in rhesus monkeys infected with simian immunodeficiency virus of macaques

The CD4 molecule is a high-affinity cell-surface receptor for the human immunodeficiency virus (HIV-1) and a soluble truncated form of CD4 produced by recombinant DNA technology is a potent inhibitor of HIV-1 replication and HIV-1-induced cell fusion in vitro. Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVMAC), a virus closely related to HIV-1, develop an AIDS-like syndrome, and so provide an important model for the evaluation of potential AIDS therapies. We have assessed the therapeutic effect of recombinant soluble CD4 in SIVMAC-infected rhesus monkeys. Virus was readily isolated from peripheral blood lymphocytes and bone marrow cells of these animals before starting treatment with soluble CD4, but became difficult to isolate soon after treatment had begun. Moreover the diminished growth of both granulocyte-macrophage and erythrocyte progenitor colonies from the bone marrow of these monkeys rose to normal levels during treatment. These findings indicate that soluble CD4 could prove valuable in the treatment of AIDS.     

Transfer of a functional human immune system to mice with severe combined immunodeficiency

The pressing need for a better experimental system for AIDS research has brought into sharp focus the shortcomings of available animal models and the practical and ethical limitations of studies of immune responses and viral pathogenesis in humans. Current studies of the human immune responses are limited to relatively restrictive in vivo experiments and several in vitro systems that, although useful, allow only short-term studies and support responses to a few antigens. Neither model is particularly amenable to studies of the pathogenesis of diseases of the immune system. We report here that injection of human peripheral blood leukocytes (PBL) can result in the stable long-term reconstitution of a functional human immune system in mice with severe combined immunodeficiency (SCID). Human PBL transplanted to SCID mice increase in number and survive for at least six months; reconstituted mice show spontaneous secretion of human immunoglobulin and a specific human antibody response is induced following immunization with tetanus toxoid. All of the major cell populations present in PBL are found in the lymphoid tissue and blood of SCID recipients, although the relative proportions of B cells, T-cell subsets and monocytes/macrophages in long-term recipients differ from those found in normal PBL and, in mice transplanted with 50 x 10(6) or more PBL from Epstein-Barr virus (EBV)-seropositive donors, EBV-positive B-cell lymphomas often develop. Our results suggest that xenogeneic transplantation of human lymphoid cells into SCID mice may provide a useful model for the study of normal human immune function, the response of the immune system to pathogenic agents and early events in lymphomagensis.     

Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barr virus.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been known for some time, but the precise role of EBV in this cancer is poorly understood, due partly to the lack of an in vitro system for studying NPC cells and the effect of EBV on epithelial cells. Biopsies of NPC tumours have revealed expression of the EBV latent membrane protein (LMP) in 65% of cases, suggesting that in at least some NPC tumours LMP may contribute to cell transformation. Here we address the question of the effect of LMP expression on epithelial cells. Transfection of an immortalized, non-tumorigenic keratinocyte cell line (RHEK-1) with the LMP gene causes a striking morphological transformation: the originally flat, polygonal colonies change to bundles of spindle-shaped cells that form multilayer foci, and cytokeratin expression is down-regulated. Our results suggest that LMP expression may be an important causal factor in the development of NPC.     

Absence of expression of OKT8 antigen on cultured human, calf and rat oligodendrocytes

Monoclonal antibodies which recognize human suppressor T cells (OKT8) have been reported by Oger and co-workers to bind to cultured sheep oligodendrocytes. These authors speculated that an immune response directed at determinants shared by suppressor lymphocytes and oligodendrocytes could explain the decrease in both circulating blood suppressor T cells and oligodendrocytes in patients with multiple sclerosis. In view of the vital issue of potential cross-reactivity between oligodendrocytes and lymphocytes, we studied the binding of viable cultured calf, rat and human oligodendrocytes using monoclonal antibodies to human T cells and monocytes. We report here that we were unable to confirm the presence of shared determinants among oligodendrocytes and any leukocytes, including human T cells or monocytes. As the reported observations of lymphocyte-oligodendrocyte shared determinants could not be identified in three other species, including man, we found no evidence to support the hypothesis that such shared determinants are of importance in the pathogenesis of multiple sclerosis.     

Phorbol ester and Epstein-Barr virus dependent transformation of normal primary human skin epithelial cells

Substantial evidence has implicated Epstein-Barr virus (EBV) in the aetiology of two human neoplasms, nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma. This is supported by the presence of high antibody titres to EBV early antigen and virus capsid antigen, as well as antibody to two viral-associated enzymes, DNase and DNA polymerase. Patients with NPC, particularly the undifferentiated form, are commonly found to have EBV DNA in the tumour. Ito and others have presented strong epidemiological evidence that phorbol esters are related to the unusual geographic distribution of NPC in southeastern regions of China. There appears to be a close link between the widespread EBV infection of the Asian population and the distinct regional distribution in China of plants that produce diterpene ester. Naturally occurring phorbol esters are produced by plants of the Euphorbiaceae and Thymelaeaceae, which are used as traditional herbal medicines. Although it has been established that EBV can infect epithelial cells isolated from NPC as well as certain normal epithelial cells, there has been no in vitro evidence that EBV induces neoplastic transformation in normal human epithelial cells with or without exposure to phorbol esters. We report here evidence that transformation of normal human epithelial cells results from exposure to infectious EBV and that transformation is dependent on the presence of phorbol esters.