Differentiation Antigens of Human Hemopoietic Cells: Patterns of Reactivity of Two Monoclonal Antibodies1
Total Page：16
File Type：pdf, Size：1020Kb
[CANCER RESEARCH 43, 4812-4815, October 1983] Differentiation Antigens of Human Hemopoietic Cells: Patterns of Reactivity of Two Monoclonal Antibodies1 Silvana Passano,2 Lisabianca Bùttero, Jeffrey Faust, Massimo Trucco, Antonio Palumbo, Luigi Pegoraro, Beverly Lange, Colette Brezin, Jannie Borst, Cox Terhorst, and Giovanni Rovera The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 [S. P., L. B., J. F., M. T., A. P., G. R.]; The Institute of Internal Medicine, University of Turin, Italy [L. P.]; Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [B. L); and The Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [C. B., J. B., C. T.] ABSTRACT terized the surface antigens recognized by 2 monoclonal anti bodies generated by immunizing mice with AML cells. These Two mouse anti-human monoclonal antibodies (S3.13 and antibodies are reactive with a large number of leukemic cell lines S5.7) raised against cells of acute myelogenous leukemia were and have a complex distribution on hemopoietic cells. found to react with antigens expressed on the surface of subsets of monocytes and lymphocytes. MATERIALS AND METHODS S3.13 precipitates a peptide of M, 29,000, and S5.7 precipi tates a peptide of M, 20,000 present on the surface of all the cell Hemopoietic Tissues. Normal peripheral blood and marrow for these types tested. These two surface antigens were distributed on investigations were obtained from healthy volunteer donors and ap proved by the Committee for the Protection of Human Subjects of The discrete subpopulations of normal hemopoietic cells. The anti Wistar Institute. bodies reacted with all (S5.7) or a subpopulation (S3.13) of Mononuclear cells were separated by FicolhHypaque (D = 1.078) peripheral blood T-lymphocytes, and with a subset of monocytes. gradient centrifugation (4), and the cells at the interface were suspended Both antibodies reacted with bone marrow blast cell progenitors in PBS and incubated with the monoclonal antibodies as described below. of the myelomonocytes and erythroid lineage. S5.7 also reacted Total peripheral blood leukocytes were prepared as described previously with non-T-lymphocytes and with cells of the eosinophilic lineage. (7, 20). Thymus tissue was obtained at autopsy within 4 hr of death. Both antigens disappeared from the cell surface during normal Cell Lines. HL-60, ML3, and KG1 (AML); THP1 (monocytic leukemia); myeloid and erythroid differentiation. Thus, these monoclonal U937 (histiocytoma); K562 (chronic myelogenous leukemia in blast crisis); Daudi and Raji (Burkitt's lymphoma); Jurkat, HPB, and HUT 78 (T-ALL); antibodies define the molecular characteristics and the cellular and Nairn 1 (non-B- and non-T-ALL) are established human cell lines distribution of two differentiation antigens present on cells of the maintained in our laboratory as described previously (16, 20). The ML3 hemopoietic lineage. cell line (19), used as an immunogen for one of the fusions, was established from the peripheral blood of a 24-year-old male with acute INTRODUCTION myelogenous leukemia. ML3 cells have the morphological and histc- chemical markers of the myeloid lineage at the promyelocyte stage. P3- Monoclonal antibodies (13) directed against antigens present x63-Ag8.653 mouse myeloma line (10) and the nonhemopoietic cell lines on hemopoietic cells are being utilized to characterize the distri described in the text were grown in Roswell Park Memorial Institute bution of differentiation antigens on normal and leukemic cells Medium 1640 containing 10% fetal bovine serum and incubated in a (23). Lineage-specific differentiation antigens have been defined humidified 5% CCvhumidified atmosphere at 37°. in several cases (12, 22), but it is often the case that the Immunization and Hybridization. Two 6-month-old female BALB/c distribution of surface antigens present on normal and leukemic mice were given i.p. injections of 2 x 107 ML3 human promyelocytic cells is not limited to a single lineage or to a specific cell leukemia cells or bone marrow cells from a patient with AML. The mice differentiation stage. For example, DR surface antigen (a dimer were given i.v. injections 7 days later with an equal number of cells. Three days after the second injection, approximately 10" spleen cells of gp33 and gp28) is expressed by B-lymphocytes, monocytes, were fused with 2 x 107 P3-x63-Ag8.653 mouse myeloma cells as myeloid, erythroid, and B-lymphocyte progenitor cells and by a fraction of myeloid leukemias and ALLs3 (1, 8, 23). The reasons described previously (16, 20). Selection of Monoclonal Antibodies. The hybrid clones reactive with underlying such a complex distribution are not yet understood the cells used for immunization were identified in radioimmunoassay by but probably reflect the peculiar and specific function of this testing the supernatant present in the colony-containing wells (16, 20). antigen. All clones that reacted with the leukemic cells were further screened for A knowledge of distribution of the various molecules on the specificity by flow cytofluorimetric analysis (see below). Two monoclonal surface of normal and leukemic hemopoietic cells will be of value antibodies reacted strongly with leukemic cells. These monoclonal anti in understanding lineage interrelationships and may help to shed bodies were identified as S3.13 (produced using AML cells from a patient) some light on the function of these molecules. We have charac- and S5.7 (produced using ML3 cells). Hybridoma cells (2 x 107) were injected i.p. into pristane-primed BALB/c mice to produce ascites ranging 1Supported in part by Grants CA 10815, CA 24273, CA 25875, and CA 14479 intiterfrom1:104to1:105. from the National Cancer Institute; Grant Al 17636 from the National Institute of Isotype Characterization. Immunoglobulin isotype was determined Allergy and Infectious Diseases; Grant JFCF 564 from the American Cancer Society; Grant CT 81.00306 from Consiglio Nazionale delle Ricerche; and a grant from the by immunodiffusion in Ouchterlony plates using goat anti-mouse immu- W. W. Smith Foundation. noglobulin class- and subclass-specific antibodies (Meloy Laboratories, 2 To whom requests for reprints should be addressed, at The Wistar Institute of Springfield, Va.). Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104. 3 The abbreviations used are: ALL, acute lymphoblastic leukemia; AMI. acute Flow Cytofluorimetric Analysis. Target cells were treated with 20 ¿il myeloblastic leukemia; PBS, phosphate-buffered saline; FITC, fluorescein isothto- of the antibody at a chosen dilution, incubated at room temperature for cyanate. 30 min, and washed 3 times. The cells were then incubated in 20 ¡Aof Received June 16, 1982; accepted June 28, 1983. FITC goat F(ab')2 anti-mouse F(ab')s antiserum (Cappel Laboratories). 4812 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1983 American Association for Cancer Research. Surface Antigens of Human Hemopoietic Cells Control cultures were treated with the supernatant of the parental mouse out on discontinuous vertical slab gels according to a modification of the myeloma and FITC-conjugated antibodies or with an irrelevant monoclo Laemmli procedure (14). Dried gels were subjected to autoradiography nal antibody of the same isotype. An Ortho Cytofluorograf System 50HH as described (3). connected to a Data General MT-200 microprocessor was used for analysis of the fluorescent cells. A 2-watt argon laser (488 nm) was the RESULTS source for generation of scatter signals and fluorescence activation. Fluorescence profiles were generated as histograms of number of cells Reactivity of Monoclonal Antibodies with Normal Hemopoi in each channel (y-axis) and relative intensity of the fluorescence over 200 or 1000 channels (x-axis), or as type of cells resolved by scattering etic Cells. Table 1 summarizes the reactivity of antibodies S5.7 in the x- and y-axes and number of cells (fluorescent or not fluorescent) and S3.13 with the hemopoietic cells. S3.13 (IgM class) reacted in the z-axis. with approximately 30% peripheral blood lymphocytes and with For flow cytofluorimetric analysis, 3 x 106 cells/sample were used. a fraction (<20%) of monocytes. The fluorescence intensity was Marrow and peripheral blood subpopulations were identified on the basis higher in lymphocytes (mean fluorescence intensity, 57/2oo)than of right- and forward-angle scatter (7, 16). The leukemic cell line popu in monocytes (mean fluorescence intensity, ^/ioo). Sorting of lations were identified by their scattering as a homogeneous population. fluorescent bone marrow cells indicated that S3.13 reacted with For sorting experiments, 2 x 106 cells/sample were used and, after a small population (<8%) consisting of lymphocytes and blast indirect immunofluorescence labeling, they were resuspended in 1 ml of cells. S5.7 (lgG1 class) reacted with a small population of gran- PBS. ulocytes (<5% of total granulocytes) and with >90% of lympho For both cytofluorimetric and sorting analysis, the threshold for fluo cytes and of monocytes (mean fluorescence intensity, 47/2ooand rescence intensity was established at the level at which 99% of the total 51/2oo,respectively). Cell sorting analysis of the fluorescent cells cell population treated with control supernatant from the parental mye loma cell line and FITC-conjugated anti-mouse antibody were negative. in the bone marrow (about 12% of the population) indicated that Sorting was performed