Human cell lines containing Epstein-Barr virus but distinct from the common B cell lymphoblastoid lines.

Abstract

In the majority of the lymphoblastoid cell lines obtained by transformation of human lymphocytes with Epstein-Barr virus (EBV), the cells are highly variable in shape and they grow in clumps in stationary cultures (1). They express many characteristics of normal B lymphocytes (2, 3). In particular, synthesis of immunoglobulin (Ig) has been the unique feature by which the B cell origin of these lymphoblastoid lines has been ascertained. In the present studies, a number of unusual lymphoblastoid lines were obtained with growth characteristics and cell morphology markedly different from those displayed by the common lymphoblastoid cell lines of the B cell type. These unusual cell lines were found to contain the EBV genome, but immunoglobulin synthesis was not demonstrable in any of them.MATERIALS AND METHODS Patients, Cell Preparation, Cell Culture, and Initiation of Cell Lines. Patients with various primary hematological malignancies, immunodeficiencies, and rheumatological disorders were from either the Rockefeller University Hospital or the Memorial Sloan-Kettering Cancer Center. Bone marrow samples, which were dispersed with sterile needles and peripheral blood, were subjected to Ficoll/Hypaque gradient centrifugation. Cell preparations from which the majority of the red cells and mature granulocytes were depleted were obtained from the interphase.The culture medium was RPMI-1640 (Microbiological Associates, Bethesda, MD) with L-glutamine supplemented with 2 mM additional glutamine, penicillin at 100 units/ml, and streptomycin at 100 ,g/ml (GIBCO). Fetal calf serum (lot P60512, Reheis, Phoenix, AZ) was used at 20% at the beginning of the cultures and later decreased to 10%. The source of EBV was culture supernatants from marmoset cell line B95-8 (4). Four-day culture supernatants from the B95-8 cell line were filtered through a 0.S-,m filter (Nalge, Rochester, NY) and used without storage. Cell lines were initiated in 16-mm tissue culture plates (FB-16-24-TC, Linbro, Hamden, CT) with 2 X 106 cells in 1 ml of medium. In some cultures, 1 ml of filtered B95-8 culture supernatant was added to the wells. Cultures were kept at 370C and 5% CO2 in a humid air atmosphere and were fed twice weekly. After the establishment of cell lines, the cells were split 3:1 every third day. Seed samples of freshly established cell lines were cryopreserved in RPMI-1640, 15% fetal calf serum, and 10% dimethyl sulfoxide in liquid nitrogen.Immunofluorescence, Hemagglutination, and Anti-Ig Rosetting Reactions. F(ab')2 fragments of affinity-columnpurified antibodies specific for IgM, IgG, IgA, and K and X light chains were used. Preparation of these antibodies was detailed elsewhere (5). Rhodamine-conjugated F(ab')2 fragments with various specificities were used for immunofluorescence. Surface and intracellular immunofluorescent staining for Ig was performed as described (6). Hemagglutination and hemagglutination inhibition was carried out as described (6). For dnti-Ig rosetting reactions, F(ab') fragments of the purified antibodies were ...

Physiology and pathology of rheumatoid factors

“…There is suggestive evidence that primarily the more mature B cells are induced by pokeweed mitogen. On the contrary, sensitivity to Epstein-Barr viral infection probably develops very early in B cell ontogeny, perhaps even prior to the appearance of surface immunoglubulin [18]. Thus, purified B cells from umbilical cord blood respond vigorously to Epstein-Barr virus, but poorly to pokeweed mitogen, even in the presence of an accessory source of mature T cells [31,40].…”

Induction of HLA-DC/DS (LEU 10) antigen expression by human precursor B cell lines.

“…By immunofluorescence, Josh 7 and Reh did not make any Ig whereas Nalm 12 made only cytoplasmic t~ chains. These cell lines represent human precursor and pre-B cell lines (11,12). Immunofluorescence studies as shown in Fig.…”

“…Human non-T ALL cell lines Reh and Nalm 12 (11) were kindly provided by Dr. P. Ralph of the Memorial Sloan-Kettering Cancer Center. Cell line Josh-7 was derived from a patient with X-linked agammaglobulinemia (12). It has little detectable Ig and is HLA-DR ÷, CALLA +, and TdT-.…”

Production of tumor necrosis factor/cachectin by human B cell lines and tonsillar B cells.

“…cell-depleted human bone marrow cells as described (9). These cells express: WIH8, y' k`; WIH10, a'x' ; WIH14, a'x' ; WIH20, la'x' ; WIH67, y' X' ; and KAW15, y' X' .…”