Multiple lymphoblastoid cell lines have been derived from two patients with chronic lymphocytic leukemia with an associated monoclonal immunoglobulin (Ig) band. Idiotypic antisera raised against the monoclonal serum Ig bands were shown to be specific for the membrane Ig of the patients' leukemic cells. The idiotypic determinants in these patients thereby constitute tumor-specific antigens. Surface and intracellular immunofluorescence studies utilizing these idiotypic antisera were used to identify the cell lines of leukemic origin. These studies showed that certain cell lines from each patient were derived from the leukemic cells while other cell lines were derived from residual normal B lymphocytes. The leukemic cell lines were variable and contained different percentages of lymphoid cells with the idiotype specific membrane Ig and, in addition, different percentages of plasma cells with intracellular Ig of the same specificity. Specific Ig synthesis was also demonstrated by hemagglutination-inhibition analysis of cell line supernatants. Aside from Ig specificity, no differences have been found between the leukemic cell lines and those derived from normal cells. One of the leukemic cell lines was cloned in soft agarose. All the clones were shown to be of leukemic origin.

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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 ...

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