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.     

Transformation of NIH 3T3 cells by a human c-sis cDNA clone

The mechanism of leukaemogenic transformation by human T-cell leukaemia/lymphoma virus (HTLV), a retrovirus implicated in the aetiology of certain adult T-cell leukaemias and lymphomas, is unknown but is conceivably associated with the expression of the cellular analogues of retroviral oncogenes. The HUT-102 cell line, derived from a cutaneous T-cell lymphoma and infected with HTLV, expresses several cellular oncogenes. It is unusual among haemopoietic cell lines in that one of these is c-sis, the gene from which the oncogene v-sis of the simian sarcoma virus was derived, and perhaps the gene for platelet-derived growth factor (PDGF). To explore the possible role of c-sis expression in HTLV-induced disease, we have obtained cDNA clones of c-sis from HUT-102 cells. Here we describe two such clones and report that one of them transforms NIH-3T3 cells. This is the first example of transformation of NIH-3T3 cells by a human onc gene other than c-ras or Blym, as well as the first demonstration of transformation by a human cDNA clone.     

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.     

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.     

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.     

Identification of reciprocal translocation sites within the c-myc oncogene and immunoglobulin mu locus in a Burkitt lymphoma

The association between certain human tumours and characteristic chromosomal abnormalities has led to the hypothesis that specific cellular oncogenes may be involved and consequently 'activated' in these genetic recombinations. This hypothesis has found strong support in the recent findings that some cellular homologues of retroviral onc genes are located in chromosomal segments which are affected by specific tumour-related abnormalities (see ref. 4 for review). In the case of human undifferentiated B-cell lymphoma (UBL) and mouse plasmacytomas, cytogenetic and chromosomal mapping data have identified characteristic chromosomal recombinations directly involving different immunoglobulin genes and the c-myc oncogene (for review see refs 5, 6). In UBLs carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24) which is translocated to the immunoglobulin heavy-chain locus (IHC) on chromosome 14. Although it is known that the chromosomal breakpoints can be variably located within or outside the c-myc locus and within the IHC mu (refs 9, 11) or IHC gamma locus, the recombination sites have not been exactly identified and mapped in relation to the functional domains of these loci. We report here the identification and characterization of two reciprocal recombination sites between c-myc and IHC mu in a Burkitt lymphoma. Nucleotide sequencing of the cross-over point joining chromosomes 8 and 14 on chromosome 14q--shows that the onc gene is interrupted within its first intron and joined to the heavy-chain mu switch region. This recombination predicts that the translocated onc gene would code for a rearranged mRNA but a normal c-myc polypeptide.     

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.     

Mitotic EBNA-positive lymphocytes in peripheral blood during infectious mononucleosis

Infectious mononucleosis (IM) is usually a benign lymphoproliferative disease caused by Epstein-Barr virus (EBV). Although EBV induces a state of continuous proliferation in infected B lymphocytes in vitro, the most prominent lymphoproliferation during IM is of activated, or atypical, T lymphocytes presumably responding to the virus or virus-infected cells. However, EBV genome-carrying cells are known to be circulating during IM, as cultured peripheral blood leukocytes from patients with the disease give rise to continuous lymphoblastoid cell lines, each cell of which contains the EBV genome and expresses the EBV determined nuclear antigen (EBNA). The proposal that EBV-infected cells in IM blood are not endowed with enhanced growth potential but are merely latently infected is supported by demonstrations that cells infected in vivo enter a viral replicative cycle when placed in vitro and that most cell lines derived from cultured lymphocytes of IM patients are infected by virus released in vitro. However the cells could also be capable of proliferation in vivo, since virus production and transformation are not mutually exclusive properties of EBV-transformed cells. Recently, EBNA has been detected in a very small fraction of peripheral blood lymphocytes of IM patients after T cells were first removed and this has been interpreted to indicate that cell transformation occurs in vivo during IM. The isolation of colonies of EBNA-positive cells from IM blood leukocytes cultures in soft agar suggests that at least some infected cells are capable of direct outgrowth into transformed cells. We report here direct evidence that circulating EBV-infected cells exhibit increased growth properties during IM.     

Nonspecific integration of the HTLV provirus genome into adult T-cell leukaemia cells

Human T-cell leukaemia virus (HTLV), previously also reported as ATLV, is a recently identified retrovirus which is closely associated with adult T-cell leukaemia (ATL) endemic in southwestern Japan and the Caribbean. Determination of the total nucleotide sequence of the HTLV genome has revealed no typical onc gene acquired from the cellular sequence. Screening of the HTLV provirus genome in tumour cells has shown that in all cases of ATL examined, the primary tumour cells contained the provirus genome and were monoclonal with respect to the integration site of the provirus. These findings suggest that ATL leukaemogenesis may be due to insertional mutagenesis in which the provirus genome is integrated into a specific locus on the chromosomal DNA and then activates an adjacent cellular onc gene, a mechanism already demonstrated in avian lymphoma and erythroblastosis induced by avian leukosis viruses. A common site of HTLV provirus integration in leukaemic cells among some ATL patients was reported by Hahn et al. but subsequently retracted. However, this retraction does not imply the random integration of the proviruses. Independently, we have been testing this insertional mutagenesis model in ATL and report here that the provirus did not have a common locus of integration in 35 ATL patients and did not integrate on the same chromosome in 2 ATL patients.     

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.     

Epidemiological evidence for causal relationship between Epstein-Barr virus and Burkitt's lymphoma from Ugandan prospective study.

Results from a prospective sero-epidemiological study initiated in Uganda in 1971 indicate that children with high antibody titres to Epstein-Barr virus structural antigens are at high risk of developing Burkitt's lymphoma. These findings strongly support a causal relationship between the Epstein-Barr virus and Burkitt's lymphoma but suggest that the oncogenic potential of the virus is realised only in exceptional circumstances.     

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.     

Epstein-Barr virus-positive Burkitt's lymphoma cells not recognized by virus-specific T-cell surveillance

The pathogenesis of Epstein-Barr (EB) virus-positive Burkitt's lymphoma (BL) appears to involve the combined actions of virus-induced B-cell proliferation, and a rare chromosomal translocation juxtaposing c-myc and immunoglobulin gene loci in a single B cell; holoendemic malarial infection in some way facilitates the oncogenic process. Outgrowth of the EB virus-positive tumour suggests either breakdown or evasion of those immune controls, in particular cytotoxic T-cell responses against the virus-induced lymphocyte-detected membrane antigen LYDMA, which limit virus-infected B-cell numbers in healthy virus carriers. Immunosuppression, such as that which malarial infection may induce, cannot itself be a sufficient explanation in this regard since our studies have identified a number of BL patients who retain detectable LYDMA-specific T-cell surveillance. The present work shows that in many cases of virus-associated BL, the emerging malignant clone is insensitive to such surveillance. Several EB virus-positive BL cell lines, recently established in vitro and expressing the class I histocompatibility locus antigens (HLAs) which restrict cytotoxic T-cell function, were not killed by HLA-matched LYDMA-specific effector populations in assays where the EB virus-positive lymphoblastoid cell line (LCL), derived from normal B cells of the same patient, sustained high levels of lysis.     

Homology of human T-cell leukaemia virus envelope gene with class I HLA gene

Human T-cell leukaemia virus (HTLV), first isolated in the United States from a patient with cutaneous T-cell lymphoma, is a unique horizontally transmitted retrovirus which is highly associated with certain adult T-cell malignancies. Also, HTLV can be transmitted in vitro to cord blood T-lymphocytes. In the accompanying paper it was shown that all T cells producing HTLV, whether cultured from infected persons or infected in vitro, bind a monoclonal antibody (4D12) which recognizes an epitope shared by certain cross-reactive class I major histocompatibility antigens. This antigen may account for the extra HLA-A and -B specificities detected in HTLV-infected cells using alloantisera. Because of the unusual findings of apparently inappropriate HLA antigens in HTLV infected cells, we had previously looked for rearrangement of class I-related genes in HTLV infected cells but failed to find any. Here, using molecular clones of HTLV and human major histocompatibility antigen DNA, we have shown 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.     

Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2

The putative oncogene bcl-2 is juxtaposed to the immunoglobulin heavy chain (Igh) locus by the t(14;18) chromosomal translocation typical of human follicular B-cell lymphomas. The bcl-2 gene product is not altered by the translocation, but its expression is deregulated, presumably by the Igh enhancer E mu. Constitutive bcl-2 expression seems to augment cell survival, as infection with a bcl-2 retrovirus enables certain growth factor-dependent mouse cell lines to maintain viability when deprived of factor. Furthermore, high levels of the bcl-2 product can protect human B and T lymphoblasts under stress and thereby confer a growth advantage. Mice expressing a bcl-2 transgene controlled by the Igh enhancer accumulate small non-cycling B cells which survive unusually well in vitro but do not show a propensity for spontaneous tumorigenesis. In contrast, an analogous myc transgene, designed to mimic the myc-Igh translocation product typical of Burkitt's lymphoma and rodent plasmacytoma, promotes B lymphoid cell proliferation and predisposes mice to malignancy in pre-B and B lymphoid cells. Previous experiments have suggested that bcl-2 can cooperate with deregulated myc to improve in vitro growth of pre-B and B cells. Here we describe a marked synergy between bcl-2 and myc in doubly transgenic mice. E mu-bcl-2/myc mice show hyperproliferation of pre-B and B cells and develop tumours much faster than E mu-myc mice. Suprisingly, the tumours derive from a cell with the hallmarks of a primitive haemopoietic cell, perhaps a lymphoid-committed stem cell.