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A Human Thymus-Leukemia Antigen Defined by Hybridoma Monoclonal

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A Human Thymus-Leukemia Antigen Defined by Hybridoma Monoclonal Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6552-6556, December 1979 Immunology A human thymus-leukemia antigen defined by hybridoma monoclonal antibodies (acute lymphocytic leukemia/cell surface/tumor antigens) RONALD LEVY, JEANETTE DILLEY, ROBERT I. Fox, AND ROGER WARNKE The Howard Hughes Medical Institute Laboratories, and the Departments of Medicine and Pathology, Stanford University Medical Center, Stanford, California 94305 Communicated by Henry Kaplan, July 9, 1979 ABSTRACT A series of mouse hybridomas producing mo- MATERIALS AND METHODS noclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Human Cells. The leukemia cells used for immunization and Among 1200 primary cultures, 60 produced an antibody that for screening of antibodies were derived from the peripheral could distinguish between the immunizing leukemia cells and blood of a child (Dom) with T-cell ALL. The patient had a an isologous B lymphoblastoid cell line. Of these, two produced mediastinal mass and a blood lymphoblast cell count of 460,000 an antibody that detects an antigen expressed preferentially on ALL cells and on a subpopulation of normal cells found in the per mm3. Greater than 95% of these blast cells formed heat- cortex of the thymus. Other normal human lymphoid cells from stable sheep erythrocyte rosettes (7). Leukemia cells from patient lymph nodes, spleen, bone marrow, and peripheral blood express Dom and a series of other patients were purified from peri- only low levels of this antigen. High levels of this "thymus- pheral blood or bone marrow or Ficoll-Hypaque sedimenta- leukemia" antigen were found on T-ALL cells, T-ALL-derived tion (8) and stored in 10% dimethyl sulfoxide under liquid N2. cell lines, and some "null" ALL cells. By contrast, B-cell leuke- A B-cell LCL from patient Dom (LCL-Dom) was established mias, B lymphoblastoid cell lines, and normal and malignant myeloid cells contain either low or undetectable amounts of this by Henry Kaplan. This cell line is composed of a polyclonal antigen. The thymus-leukemia antigen has been isolated from mixture of K and X immunoglobulin-bearing cells (9) and it the membranes of leukemia cells by detergent solubilization contains the Epstein-Barr nuclear antigen (10) (Henry Kaplan, and subsequent immunoprecipitation with the monoclonal personal communication). The LCL-Dom cells were grown and antibody. Preliminary biochemical characterization shows the maintained in Dulbecco's modified Eagle's medium (high antigen to be associated with a polypeptide of Mr -28,000. glucose) (GIBCO), containing penicillin (100 units/ml), gluta- A major advance in cell surface and tumor immunology has mine (2 mM), and 15% fetal calf serum. Other cell lines used been the introduction of techniques for the production of ho- in these studies include the T cell lines MOLT-4, 8402, and mogeneous monoclonal antibodies. It is now possible to sort HSB2 and the B cell lines 8866, 8392, and SB. Their origins and through a complex mixture of antigens, such as cell surface characteristics have been described (11-13). Normal human molecules, with probes that see one antigen at a time. These thymus glands, lymph nodes, and spleens were obtained from techniques are especially applicable to the study of human cell fresh surgical specimens. surface antigens, particularly in the search for human tumor- Immunization Schemes. BALB/c mice were immunized specific antigens. Monoclonal antibodies can be obtained by and 1 week later were given an intraperitoneal booster with 107 the Klinman technique, which involves in vivo cloning of B cells glutaraldehyde-fixed (4) ALL-Dom cells. Three months later, by adoptive cell transfer and subsequent in vitro spleen frag- they were given an intravenous booster with another 107 ment culture (1). By using this method, we demonstrated that ALL-Dom cells. Three days later, 2 X 107 spleen cells from the mouse has within its antibody repertoire the capacity to these animals were transferred intravenously to syngeneic ir- distinguish among closely related human cell surface antigens, radiated (600 rads) mice along with another 107 ALL-Dom including HLA polymorphisms (2), B- and T-cell determinants cells. The recipient mice had been previously hyperimmunized (3), and tumor-associated antigens (4, 5). However, this tech- either with normal peripheral blood lymphocytes (PBL) (group nique was ultimately limited by the amount of antibody ob- I), LCL-Dom (group II), or nothing (group III, control). Hy- tainable from each spleen fragment culture. With the intro- bridomas were prepared from the spleen cells of each recipient duction of the hybridoma technique of Kohler and Milstein (6), group 1 week after transfer. it became possible to obtain monoclonal antibodies in large Cell Fusion and Culture. Spleen cells were fused with amounts. NSI/1-AG 4 cells (14) at a cell ratio of 2:1, respectively, by using In the present study, we generated a panel of individual 38% polyethylene glycol (Baker 1540) (5). After fusion, the cells hybridomas from mice immunized to human acute lympho- were resuspended in Kennet's modification of Dulbecco's cytic leukemia (ALL) cells. These antibodies were screened for medium containing hypoxanthine, aminopterin, and thymidine their ability to discriminate between the leukemia cells and an (15) and dispensed into 96-well tissue culture trays (Linbro) at isologous B lymphobastoid cell line (LCL). Among the dis- 1-2 X 105 cells per well along with 5 X 105 normal spleen cells. criminator antibodies, two define an antigen that is preferen- The medium was changed twice during the first 2 weeks to tially expressed on ALL cells and on a subpopulation of normal remove antibody released by unfused spleen cells and then was thymocytes. collected for testing during the third week. Culture fluids were sampled, with attention being paid to avoid contamination of The publication costs of this article were defrayed in part by page each fluid by its neighbors-i.e., by washing or changing of charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: ALL, acute lymphocytic leukemia; LCL, lympho- this fact. blastoid cell line; PBL, peripheral blood lymphocytes. 6552 Downloaded by guest on September 25, 2021 Immunology: Levy et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6553 pipettes between collections. These fluids were examined for the presence of antibody against the immunizing ALL-Doxn cells as well as the LCL-Dom cells by a cell-binding radcoim- munoassay (5), using purified 125I-labeled goat anti-mouse Fab fragment as a detecting reagent. Selected cultures were sub- It cloned by'plating limiting dilutions of the hybrid cells into the wells of 96-well plates along with normal mouse spleen cells. Immunofluorescence. Hybridoma antibodies were exam- ined by indirect immunofluorescence on viable cells in sus- pension, fixed cell smears, or frozen tissue sections prepared by described methods (9). A rhodamine conjugate of the same purified goat anti-mouse Fab used in the radioimmunoassay was employed. In some cases, an F(ab')2 fragment of this pu- -11 11 rified goat antibody was employed. E S S"- WNgS1E1 11S Labeling of Cell Membranes and Immunoprecipitation. Cell membranes were radio-iodinated by using lactoperoxidase and solubilized with Nonidet P-40 (Shell Chemical) as described (16). The solubilized 125I-labeled membrane preparation, 107 cell equivalents, was first subjected to a nonspecific immu- 2000 7 noprecipitation by the addition of human IgG (25 Mg) and goat anti-human Fab at equivalence. To the cleared supernatant, 4000 - 100-200 ,l of culture fluid containing either a specific mono- clonal antibody or a myeloma protein of the same class was added. This was followed by the addition of normal mouse serum (2.5 pi) and goat anti-mouse Fab (preabsorbed on human 6000 IgG-Sepharose) at equivalence. The final precipitates were solubilized in 3% sodium dodecyl sulfate/2% mercaptoetha- _ LCL nol/0.05 M Tris-HCI, pH 6.8/5% glycerol and boiled. FIG. 1. Radioimmunoassay of hybridoma culture fluids. Fluids Gel Electrophoresis. Sodium dodecyl sulfate/polyacryl- from individual culture wells were assayed for binding activity against amide gel electrophoresis (17) was performed in slab gels by two different target cells from patient Dom: ALL (Upper) and LCL using either a 5-20% gradient of acrylamide or a uniform 10% (Lower). The data from one representative 96-well plate are dis- acrylamide concentration. Gels wer'e dried and autoradio- played. graphed by using Kodak XR2 film with a Cronex Lightning Plus intensifying screen (Dupont). groups the recipients had been hyperimmunized against normal Fluorescence-Activated Cell Sorter Analysis. Lymphocytes human lymphoid antigens-either PBL (group I) or LCL-Dom were incubated with monoclonal antibody (50 Ml per 5 X 106 (group II). This maneuver was an attempt to suppress the ex- cells) at 40C in the presence of 1 mM azide. The cells were pansion of B cell clones reactive to normal human antigens washed in fetal calf serum and gently resuspended in 50 Ml of while stimulating the expansion of clones reactive to antigens fluorescent goat anti-mouse Ig antibody. The stained cells were limited to the leukemia cell. A third group of recipients was not washed again in fetal calf serum, resuspended in Dulbecco's preimmunized. Similar numbers of total antibody-producing phosphate-buffered saline, and fixed in 1% formaldehyde. hybridomas were obtained in each group. However, a moderate Analysis of staining intensity was performed by using the flu- advantage was noted for groups I and II in terms of the per- orescence-activated cell sorter as described by Loken and centage of hybrids producing antibody that discriminated be- Herzenberg (18). tween the leukemia cell and the isologous B cell line-13.8 and 9.7%, respectively, vs.
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