Continuous Lymphoid Cell Lines with Characteristics of B Cells (Bone-Marrow-Derived), Lacking the Epstein-Barr Virus Genome and Derived from Three Human Lymphomas

Klein, George; Lindahl, Tomas; Jondal, Mikael; Leibold, W.; Menezes, José; Nilsson, Kenneth; Sundström, Christer

doi:10.1073/pnas.71.8.3283

Cited by 304 publications

(122 citation statements)

References 18 publications

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“…Virus was first detected at 48 hr after transfection in one experiment and at 72 hr in the other two. Maximal titers of about 105 PFU/ml were reached after [4][5][6] days.…”

Section: Susceptibilitymentioning

confidence: 97%

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Transfection of human lymphoblastoid cells with herpes simplex viral DNA.

Miller¹,

Wertheim²,

Wilson³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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The "calcium/dimethyl sulfoxide shock" method of transfection was adapted for use in human lymphoid cell cultures. One microgram of herpes simplex virus type 1 DNA regularly initiated virus replication in four lymphoblastoid cell lines. Per 105 cells exposed to 1 pg of DNA, 0.5-5 cells formed an infectious center. The minimal infective dose of DNA was approximately 500 ng.A reliable method for transfection of human lymphocytes by DNA could have many applications in the study of lymphotropic viruses and also might be useful in experimental immunology and genetics. To initiate studies of this problem we chose a system that would maximize the chances of demonstrating expression of genetic information after introduction of naked DNA into lymphoid cells. The DNA of herpes simplex virus (HSV) was selected because one assay for expressionnamely, production of complete progeny-is simple and rapid and because several methods are already available for detection of infectious herpes DNA on monolayer tissue culture cells (1-3). The ability to assay HSV-1 DNA independently in permissive cells provided a check on the quality of the DNA and on the adequacy of the transfection technique. HSV DNA was chosen also because it is a good model for future studies with Epstein-Barr virus (EBV) DNA, a molecule of the same size.MATERIALS AND METHODS Virus Strain, Propagation, and Preparation of Viral DNA. The Lovering strain of HSV-1, originally from a fetal encephalitis case, was passed at low multiplicity three times in the AH-1 and once in the BSC-1 line of African green monkey kidney cells (4). The virus was plaque-purified in BSC-1 cells under 2% methylcellulose. Two batches of viral DNA were used: batch 4, prepared from virions propagated in BSC-1 cells; batch 6, derived from a subclone obtained in transfection of BSC-1 cells, was prepared from virions propagated in BHK-21 cells.Viral DNA was obtained from virions by the procedure described by Geelen et al. (5) for preparation of infectious cytomegalovirus DNA. The starting material for each batch of DNA was 6-20 roller bottles of BSC-1 cells. The medium was removed from confluent cultures, which were inoculated with about 2 plaque-forming units (PFU) per cell. After 1 hr the inoculum was removed and 50 ml of Eagle's medium with 2% calf serum was added to each bottle. After 40 hr, when there was extensive cytopathic effect, the culture medium was decanted and centrifuged at 4000 rpm at 4°C for 10 min in the Sorvall GSA rotor. The supernatant fluids were separated from the pellets and centrifuged for 3 hr at 18,000 rpm Lymphoid Cells. Three lymphoblastoid lines, designated X25, were derived after transformation of lymphocytes from one umbilical cord with a passage of the B95-8 strain of EBV (unpublished data); the fourth line (X50-7) originated from another umbilical cord. These lines are not spontaneous producers of extracellular EBV but transforming virus can be recovered from the lines by the technique of x-irradiation and cocultivation. All the cells have EBV nuclear ...

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“…Virus was first detected at 48 hr after transfection in one experiment and at 72 hr in the other two. Maximal titers of about 105 PFU/ml were reached after [4][5][6] days.…”

Section: Susceptibilitymentioning

confidence: 97%

“…Some of the cells in the X25-9 line adhere to plastic surfaces, but adherent cells are not found in the X25-4, X25-6, or X50-7 lines. Raji and BJAB are Burkitt lymphoma lines; Raji has the EBV genome and BJAB does not (6). Mixed mononuclear leukocytes from umbilical cord blood were prepared for culture after isolation on a Ficoll/Hypaque gradient (7).…”

mentioning

confidence: 99%

Transfection of human lymphoblastoid cells with herpes simplex viral DNA.

Miller¹,

Wertheim²,

Wilson³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Ramos cells For these studies predominantly two clones of P3HR-1 EBVconverted BJAB cells were analyzed: clone A 7 revealing faint granular EBNA fluorescence in almost every cell, and clone B 1 showing brilliant EBNA staining, but segregating also EBNA-negative cells (7). Treatment of both types of clones with the "induction cocktail" resulted in about 0.5% of cells in EA induction 3 days later.…”

Section: Induction Of Antigens In Ebv-converted Bjab Andmentioning

confidence: 99%

“…Two Epstein-Barr virus (EBV-negative tissue culture lines with bone-marrow-derived (B)-cell characteristics have been established from Burkitt lymphomas: the BJAB line from an EBV-negative African Burkitt's lymphoma (1) and the Ramos line (2) from an American tumor. Cells of both lines are readily infected by EBV, resulting in persistence of EBV DNA and in expression of Epstein-Barr nuclear antigen (EBNA) (2)(3)(4).…”

mentioning

confidence: 99%

Transient induction of a nuclear antigen unrelated to Epstein-Barr nuclear antigen in cells of two human B-lymphoma lines converted by Epstein-Barr virus.

Fresen¹,

Hausen²

1977

Proc. Natl. Acad. Sci. U.S.A.

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In the course of our studies on induction of early antigen (EA) and viral capsid antigen (VCA) in EBV-converted BJAB and Ramos cells and their respective clones and subclones, it became apparent that certain sera reacted with a distinct proportion of nuclei from EBV-converted lines after iododeoxyuridine induction followed by methanol fixation a few days later. No nuclear fluorescence was observed after acetone fixation or before or after induction and methanol fixation of noninfected BJAB, Ramos, P3HR-1, and Raji cells. The following is an account of these studies aiming at the characterization of the nuclear antigen and associated serological response in various groups of patients. MATERIALS AND METHODSCells. BJAB and Ramos cells were kindly provided by George Klein, Stockholm. EBV infection and conversion experiments as well as cloning of EBNA-positive sublines has been described before (4,7). P3HR-1 and Raji cells were furnished by Werner Henle, Philadelphia. B 95-8 cells were a gift by George Miller, New Haven. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), gentamycin, penicillin, and streptomycin. They were kept in Erlenmeyer flasks and fed once every week.Induction Experiments. Induction experiments were performed according to Hampar et al. (8). Approximately 107 cells were sedimented and resuspended in regular tissue culture medium containing in addition 20,ug/ml of iododeoxyuridine, 0.4 ug/ml of aminopterin, and 14,ug/ml of hypoxanthine. After 24 hr of incubation at 370, the cells were washed twice with RPMI 1640 and then resuspended in fresh medium without the "induction cocktail." They were harvested at various intervals thereafter and examined for specific immunofluorescence.EA induction by P3HR-1 Virus. Infection of cells by P3HR-1 virus was performed according to Henle et al. (9) and has been described in detail (7). Differpntiation of R-and D-subspecificities of the EA complex followed the procedures described by Henle et al. (10).Immunofluorescence Studies. Air-dried smears of cells were fixed either in acetone or in methanol for 3 min at -20'. Indirect immunofluorescence was performed according to Henle and Henle (11). Goat antiserum to human IgG was purchased from Behringwerke, Marburg, and used at a standard dilution of 1:40. The detailed procedure of anticomplement-immunofluorescence (4)

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“…The cells carry surface-bound immunoglobulin, EBV receptor (2), but do not harbor any detectable EBV DNA genome (3) or EBV-determined nuclear antigen (EBNA) (4). By infecting this cell line with transforming EBV derived from the B95-8 strain, a permanently EBV-converted subline was established (5).…”

mentioning

confidence: 99%