Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Re-exposure of human lymphoblastoid cell lines to Epstein-Barr virus

Human lymphoblastoid lines of various origins which harbour Epstein-Barr virus (EBV)-specific nucleic acid were re-exposed to EBV. Following infection, cells of the non-virus-producing lines, Raji and S 95, predominantly synthesized EBV-specific early antigens (EA), whereas only a small percentage of cells revealed viral capsid antigens (VCA). In Raji cells, the number of VCA-producing cells was paralleled by the percentage of virus-specific DNA-synthesizing cells. In S 95 cells, however, viral DNA-synthesizing cells exceeded the number of VCA-producing cells by a factor of more than 10. Induction of EA in Raji cells was dose-dependent and inversely related to cell growth. Irradiation of the virus by ultraviolet light prior to infection led to reduced infectivity. This reduction seemed to follow single-hit kinetics. Raji cells, previously re-exposed to EBV, showed reduced EA induction after re-infection with EBV, as compared to Raji control cells not previously exposed. Of 10 lines which spontaneously synthesize EBV-specific antigens, seven lines proved to be refractory to re-infection, whereas three were as susceptible as the Raji and S 95 controls. From three of the refractory lines infectious virus could be recovered from the culture medium prior to infection. These results permit the following interpretations: (1) the response of human lymphoblastoid cells after re-infection with EBV results from the infecting virus and not from stimulation of endogenous genomes; (2) cells demonstrating EA synthesis ultimately die; (3) re-exposure to EBV increases the resistance to re-infection of the surviving cells; and (4) cell lines producing infectious EBV are refractory to re-infection. It is suggested that the spontaneous synthesis of infectious virus favours the selection of resistant cells.     

Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.     

Combined effect of the extracts from Croton tiglium,Euphorbia lathyris or Euphorbia tirucalli and n-butyrate on Epstein-Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells

The combined usage of n-butyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) or the oily extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli exerted a marked effect on induction of Epstein-Barr virus (EBV)-associated early (EA) and viral capsid (VCA) antigens in EBV genome-carrying human lymphoblastoid cell lines. In producer P3HR-1 cells, the enhancing effect of the 2 components was additive both for EA and VCA, while in non-producer Raji cells, a synergistic increase of EA was observed. The possible implication of these findings relating to the cause of EBV-associated diseases is discussed.     

Differential induction of Epstein-Barr virus-related antigens in heterokaryon cultures.

Different paterns of induction of Epstein-Barr virus (EBV)-related antigens were observed in heterokaryons produced by Sendai virus-mediated fusion of producer and non-producer human lymphoblastoid cells with various other cell types. EBV-related early antigens (EA) and viral capsid antigen (VCA) could obviously be induced in heterokaryons between producer cells (P3HR-1 and QIMR-WIL), normally expressing these natigens at very low frequency, and human FL or HeLa cells. Positive cells were detected as early as 3 h after fusion and there often followed a rapid increase in positive cells. In contrast, in heterokaryons between non-producer cells (Raji and NC-37) and FL or HeLa cells, only EA but not VCA was induced. EA induction was also evident in fusion of human lymphoblastoid cells with monkey cells (Vero) but with mouse cells (L-M(TK-) C11D and MCB-2) no EBV induction occurred. The EBV induction in heterokaryons was significantly enhanced by 5-iododeoxyuridine treatment.     

Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.     

HTLV-III infection of EBV-genome-positive B-lymphoid cells with or without detectable T4 antigens

The infection of a number of new and established B-cell lines by human T-cell lymphotropic virus III (HTLV-III) was investigated. The B lymphocytes differed in their expression of T4 antigens detected by specific monoclonal antibodies (MAbs) and the presence of Epstein Barr virus (EBV)-DNA or antigens. The presence of the EBV genome was the only requirement for infection of B-lymphocytes by HTLV-III, although its presence did not ensure infection. Two EBV genome and T4 antigen-positive B-cell lines, lacking EBV early antigens (EA) and viral capsid antigens (VCA), could be productively infected with no induction of known EBV antigens. Two other EBV genome-positive cell lines, lacking T4, EA, and VCA could also be infected. Another genome-positive cell line (P3HR-I) that was EBV-EA, VCA-positive and produced non-transforming EBV, could also be infected by HTLV-III. However, 3 EBV genome- and T4 antigen-negative B-cell lines could only be infected with HTLV-III after successful conversion to an EBV-genome-positive state by pre-infection with EBV. Five other EBV-genome-positive B-cell lines lacking T4 antigens were not infectible with HTLV-III even after super-infection with EBV. Incomplete inhibition of the HTLV-III infection of a T4-positive (LDV-7) and a T4-negative (Craig) was obtained by preadsorption with specific MAb to T4 (OKT4A and Leu 3A). From these observations, it is not clear whether the presence of T4 antigen on the cell surface is needed for the infection of B lymphoblastoid cells; however, successful infection does depend upon the presence of the EBV genome. The mechanism of interaction of HTLV-III and EBV-infected B-cell lines permitting this infection is not fully understood. Although the clinical implications of these observations remain to be determined, it is possible that infection of EBV-positive B-cells may contribute to aberrant humoral responses and/or increased frequency of B-cell malignancies observed in HTLV-III-infected individuals.     

Syngeneic and allogeneic cell-mediated cytotoxicity against marek's disease lymphoblastoid tumor cell lines

Cell-mediated cytotoxicity (CMC) of lymphocytes obtained from chickens infected with Marek's disease (MD) virus against allogeneic MD lymphoblastoid cell lines has been reported by several research groups. Recently, we established a number of cell lines from MD tumors obtained from highly inbred chickens and characterized for major and minor histocompatibility antigens. Allogeneic versus syngeneic CMC was studied using those cell lines and lymphocytes obtained from chickens 6-8 days post infection with 5B-1, a non-oncogenic MD virus. Allogeneic cytotoxicity could be easily demonstrated, while syngeneic cytotoxicity was a rare event. However, increase of the CMC assay period from 4 to 8 h did enhance syngeneic cytotoxicity. Cold inhibition assays demonstrated that the allogeneic cytotoxicity was directed against alloantigens present on spleen lymphocytes sharing the same major histocompatibility antigens as the target cells. Cytotoxicity was not influenced by the sex of either target or effector cells or by the level of virus infectivity of the effector cells.     

Isolation of transforming and early antigen-inducing Epstein-Barr virus from nasopharyngeal carcinoma hybrid cells (NPC-KT)

The biologic activities of Epstein-Barr (EB) virus from an epithelioid-nasopharyngeal carcinoma hybrid cell line (NPC-KT) and three subclones from the NPC-KT cells were examined. Much infectious virus was released by treatment with 5-iodo-2'-deoxyuridine. All virus preparations from NPC-KT cells and the subclones were found to possess both transforming and EB virus-induced early antigen (EA)-inducing activities. The lymphoblastoid cell lines, which were established by infection of human cord-blood lymphocytes with the virus, were diploid with normal karyotypes. The cell lines were positive for EB virus-associated nuclear antigen but negative for EA and EB viral capsid antigen.     

High irradiation sensitivity of epstein-barr-virus (ebv) in lymphoblastoid-cells from patients with ataxia-telangiectasia and ebv genome-positive burkitts-lymphoma

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines derived from the peripheral blood of patients with ataxia telangiectasia (AT) and EBV genome-positive Burkitt's lymphoma (BL) were tested for expression of EBV-related lytic antigens by means of irradiation. We used 1 Gy in each experiment, according to the results of the P3HR-1 (derived from African BL) cell line. Significantly higher expression of early antigens (EA) and viral capsid antigen (VCA) was demonstrated in lymphoblastoid cell lines derived from both patients with AT and those with EBV genome-positive BL, as compared to those derived from healthy individuals. These results suggested that defective regulating mechanisms on B lymphocytes, responsible for EBV infection, may underlie for the pathogenesis of development of lymphoproliferative diseases both in patients with AT and EBV genome-positive BL.     

The establishment of lymphoblastoid lines from adult and fetal human lymphoid tissue and its dependence on EBV

Continuous human lymphoblastoid cell lines (LL), derived from lymphoid tissue or peripheral blood of 20 adults without histories of recent infectious mononucleosis at a frequency close to 100 %, were examined for the presence of Epstein-Barr virus (EBV) using immunofluorescence methods for the detection of EBV-dependent cell membrane and EB viral capsid antigens. All lines except one were found to be EBV carriers in the initial tests, but the antigens gradually disappeared in most during the course of an observation period of 4 months. Only eight lines maintained the initial percentage of antigen-containing cells. The results of the two immunofluorescence tests, performed simultaneously on each cell pool, were concordant in all instances. Sera were available from 16/20 donors. All cell donors, except one, possessed antibodies to EBV as an indication of prior EBV infections. The growth-promoting role of EBV in the establishment of LL was supported by studies with fetal lymphoid tissue cultured by the same grid method which yielded the high frequency of LL from adults. In sharp contrast to the results obtained with adult lymphoid tissue, no lines were established from 20 fetuses aged 13-20 weeks. However, when such tissue was exposed to a cell-free filtrate prepared from an EBV-carrying LL lymphoblastoid cell, lines were established in some instances. Filtrate from an EBV-negative line inoculated into parallel cultures failed to promote the establishment of LL. The results indicate that EBV infection in vivo or in vitro may be a prerequisite for the indefinite growth of lymphoblastoid cells in vitro and that EBV infections, as a rule, are not vertically transmitted.     

Epstein-barr-virus hypersensitivity of lymphocytes from patients with ataxia-telangiectasia

Ataxia telangiectasia (AT), an autosomal recessive disorder with a high incidence of lymphoreticular malignancies including Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (LPD), was investigated to assess the susceptibility to EBV infection and oncogenesis. When the patients' lymphocytes were infected with B95-8 EBV, there was a tendency toward an enhanced growth in semisolid agar, as compared with the healthy donor counterparts. Among the preparations tested, from 14 patients, 2 cell lines showed extremely high colony forming efficiency. The lymphocytes from patients with AT did not contain a large number of EBV target cells, as determined by the maximum frequency of EBV-determined nuclear antigen (EBNA) induction prior to cellular DNA synthesis. Fourteen different lymphoblastoid cell lines derived from the 14 patients with AT were then examined for their EBV inducibility and superinfectibility. By treatment with 12-O-tetradecanoyl-phorbol-13-acetate TPA) and culturing at a lower temperature of 33-degrees-C, early antigen (EA) induction occurred approximately 6-fold and 5-fold higher, respectively, as compared with the lymphoblastoid cell lines derived from healthy controls. Viral capsid antigen (VCA) was also induced significantly by TPA or culturing at lower temperature in the lines from patients with AT, but only slightly in the control counterparts. When the lymphoblastoid cells from patients with AT were exposed to P3HR-1 EBV, EA and VCA syntheses were approximately 6- and 12-fold higher, respectively, than those in the cells derived from the healthy controls. This evidence suggested B lymphocytes of patients with AT were highly susceptible to EBV infection and possibly linked to the development of EBV-induced LPD.     

Characteristics of established myeloma and lymphoblastoid cell lines derived from an E myeloma patient: a comparative study

The in vitro characteristics of two types of cell lines established from bone marrow and peripheral blood of an E myeloma patient have been compared. Both types, one with a lymphoblast morphology and the other with plasma cell/plasmablast morphology, secreted monoclonal immunoglobulins in vitro. The former produced IgGK while the latter synthesized the IgEL myeloma protein, which in a previous report was shown to be identical with the myeloma protein in vivo. The myeloma cells were difficult to establish and more stringent in their in vitro requirements than the lymphoblastoid cells. Myeloma cells could only grow in the presence of feeder cells or medium harvested from such cells, while lymphoblasts were capable of independent growth in standard media. The lymphoblastoid line was principally similar to those obtainable from normal lymph nodes (Nilsson et al., 1968; Nilsson, 1971a) and is therefore regarded as being of non-neoplastic origin. It is thus possible to obtain permanent immunoglobulin-secreting lines of non-neoplastic origin as well as myeloma lines of neoplastic origin from patients with myelomatosis. The dynamic and static morphology of the former most closely correspond to the appearance described for lymphoblasts or “immunoblasts”,i.e. lymphocytes stimulated by phyto-hemagglutinin, while myeloma cells resemble plasma cells of varying maturity. The reasons for the morphologic differences are unknown, but they were sufficiently distinctive to permit unequivocal distinction between the two types of lines.     

High-frequency establishment of human immunoglobulin-producing lymphoblastoid lines from normal and malignant lymphoid tissue and peripheral blood

Human hematopoietic tissue and lymphocytes separated from 10-20 ml samples of peripheral blood have been grown in vitro in a lens-paper and a gelatin foam (Spongostan) grid organ culture. Lymphoblastoid cell lines were established from the lymph nodes, and in one case from the spleen, of 22/23 consecutive, unselected adult individuals without manifest malignancy or infectious mononucleosis. Biopsies from 5/8 patients with malignancy were successful. The blood tines were derived from 5/10 patients with and 4/10 donors without malignancy. The very high frequency of success from normal tissue confirms the assumption made before that the spontaneous establishment of lymphoblastoid cell lines is unrelated to manifest malignancy of the donor. The results indicate that lymphoid cells with a potential for infinite proliferation (“lymphoblastoid transformation”) are present in almost all adult individuals. The Spongostan grid culture is a superior instrument to select and/or adapt these cells in vitro. All lymphoblastoid lines produced immunoglobulins. The majority started with a “polyclonal” pattern of immunoglobulin production but changed towards stable “ monoclonality “ during the course of long-term cultivation. It is suggested that lymphoblastoid lines have a polyclonal origin and that the reason for development of a monoclonal line is a selection of one cell clone either in the organ culture during establishment or in long-term culture.     

Activation of Epstein-Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells with propionic acid and with culture fluids of propionic acid-producing anaerobes

Epstein-Barr virus (EBV)-associated early (EA) and virus capsid antigens (VCA) were efficiently induced in the viral genome-carrying human lymphoblastoid cells, P3HR-1 and Raji, by the culture fluids of Propionibacterium acnes, P. avidum, P. lymphophilum and Arachnia propionica, the anaerobes which are commonly seen among the normal flora of man. The active principle for EBV-induction in the 2 cell lines was the propionic acid produced by the microbes and such activity was shown to correlate with the fatty acid content of the culture media.     

Relationship between the sensitivity of EBV-carrying lymphoblastoid lines to superinfection and the inducibility of the resident viral genome

Human, EBV-carrying lymphoblastoid lines show wide differences in their sensitivity to superinfection with EBV concentrates and also in their sensitivity to the activation of the resident viral genome by BUDR and IUDR. A significant correlation was found between sensitivity to superinfection and activation in 23 virus-receptor-positive lines. In 10 receptor-negative lines, there was no such correlation: they were resistant to superinfection since they could not adsorb the virus, but differed widely in their activatability. The findings suggest that the intracellular restrictive mechanism that limits superinfection in the receptor-positive, resistant lines, can also restrict the function of the activated genome, derived from within. Since some of the lines that were resistant to both superinfection and activation were spontaneous producers, however, it appears that the same mechanism does not necessarily affect the “spontaneous” function of the resident genome.     

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.     

Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.

Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.     

Demonstration of Marek's disease tumor-associated surface antigen in chickens infected with nononcogenic Marek's disease virus and herpesvirus of turkeys.

The presence of Marek's disease tumor-associated surface antigen (MATSA) was demonstrated on spleen cells from P-line chickens inoculated 5--6 days earlier with herpesvirus of turkeys and SB-1 (a nononcogenic Marek's disease virus). Antisera against MATSA expressed on five Marek's disease lymphoblastoid cell lines were able to recognize the MATSA present on SB-1-infected spleen cells. No viral membrane antigens and only a low incidence of viral internal antigens could be demonstrated.