Differentiation Antigens of Human Hemopoietic Cells: Patterns of Reactivity of Two Monoclonal Antibodies1

[CANCER RESEARCH 43, 4812-4815, October 1983]

Silvana Passano,2 Lisabianca BÃ¹ttero, 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 37Â°. 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 Â¿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 Â¡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

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 at a rate of 3 x 103 cells/sec. Positive and S5.7 reacted with all of the cells of the eosinophilic lineage, negative cells were separately collected into tubes containing 1 ml of promyelocytes, and blast cells. This antibody did not react with PBS supplemented with 10% fetal bovine serum. Cytocentrifuge smears mature myeloid cells or with erythroblasts. Separation of periph were stained with either Wright-Giemsa or May-Grunewald-Giemsa. At eral blood lymphocytes based on erythrocyte rosette-forming least 300 cells/slide were identified morphologically, and at least 3 ability indicated that S3.13 reacted with a subpopulation of T- separate sorting experiments on different bone marrows were performed lymphocytes (50%), and S5.7 reacted with essentially all T- and with each of the 2 monoclonal antibodies tested. non-T-lymphocytes. Sorting of the granulocytes of a patient with Preparation of Erythrocyte Rosettes. Mononuclear leukocytes ob secondary eosinophilia using antibody S5.7 indicated that the tained after FicolhHypaque gradient centrifugation of normal peripheral blood were allowed to adhere to plastic dishes for 1 hr at 37Â°.Nonad- antibody reacted with all of the peripheral blood eosinophils but herent cells were then incubated with neuraminidase-treated sheep not with neutrophils. Antibodies S5.7 and S3.13 reacted with erythrocytes for 30 min on ice and were centrifuged on a FicolhHypaque about 80 and 40%, respectively, of the thymocytes. gradient. Erythrocyte rosette-positive (pellet) and erythrocyte rosette- Reactivity of S3.13 and S5.7 with Leukemic Cell Lines. negative (interface) cells were processed for immunofluorescence as Representative T-, B-, null, and myeloblastic cell lines were described above. tested for their reactivity with S3.13 and S5.7 (Table 2). S3.13 Radiolabeling and Immunoprecipitation. Exponentially growing Jur- reacted with T-cell lines, with the non-T- non-B-cell line Nairn 1, kat and HPB cell lines or primary leukemic cells freshly obtained from an and with K562 cells derived from a chronic myeloblastic leukemia AM L patient or normal peripheral blood lymphocytes were used for patient in blast crisis but did not react with B-cell lines or with labeling experiments. Cell surface iodination with Na125lwas catalyzed by lactoperoxidase and hydrogen peroxide (18). For metabolic labeling, Table 1 the cells were washed once in Roswell Park Memorial Institute 1640 Tissue Culture Medium without L-methionine (Grand Island Biological Reactivity of monoclonal antibodies S5.7 and S3.13 with hemopoietic cells Co. Laboratories, Grand Island, N. Y.) and cultured for 14 hr at 5 x 106 antibodyS5.720-25%NegativeNegative CellsTotal cells/ml in the appropriate medium supplemented with 10% dialyzed fetal calf serum in the presence of L-^SJmethionine (400 Ci/mmol; Amer- blood"ErythrocytesGranulocytesperipheral sham/Searle Corp., Arlington Heights, III.). After labeling, cells were lysed with 1% Nonidet P-40 (Particle Data (neutrophilic) (eosinophilic)6TotalGranulocytes Laboratories, Elmhurst, III.) in 0.01 M Tris-HCI (pH 7.8):0.15 M NaCI:1 >90%>95%>95%>95%>80%Negative8-12%NegativePositiveNegative30%Negative50%15%Negative1-8%Negative>90%NegativeNegativeNegativeNegativeNegative40%

ÃŽÃ•Ã•Mphenylmethylsulfonylfluoride:ovomucoid trypsin inhibitor (0.02 mg/ lymphocytesNon-T-lymphocytesT-lymphocytesMonocytesPlateletsTotal ml) (Sigma Chemical Co., St. Louis, Mo.) (17). The lysates were centri fuged at 13,000 x g for 15 min to remove nuclei. The supernatant fluid was centrifuged for 30 min at 100,000 x g, then precleared with heat- inactivated formalin-fixed Staphylococcus aureus, and subsequently pre cleared with a preformed complex of mouse IgG and rabbit anti-mouse marrow6ErythroblastsMyeloblastsPromyelocytesMyelocytes IgG, each for 1 hr at 4Â°.Precleared lysates were incubated for 3 to 4 hr ND)CPositive(% with a preformed complex of the monoclonal antibody and rabbit anti- mouse IgG at 4Â°(18, 24). The precipitate was resuspended in 0.01 M ND)Negative>90%Negative>90%>80%S3.1312-15%NegativeNegative(% (neutrophilic)Myelocytes Tris-HCI (pH 7.8):0.15 M NaCI:1 mM phenylmethylsulfonyl fluoridertrypsin (eosinophilic)Metamyelocytes inhibitor (0.02 mg/ml) (Tris:NaCI buffer):0.05% deoxycholate sodium salt (neutrophilic)Metamyelocytes and washed on a discontinuous gradient consisting of one layer of 10% (eosinophilic)Thymocytes6Monoclonal sucrose, 0.5% Nonidet P-40 in the Tris:NaCI buffer, and one layer of 20% sucrose in the same buffer without detergents. The precipitate was then washed again in the Tris:NaCI buffer containing 0.2% Nonidet P-40 Reactivity was determined by radioimmunoassay and by cell sorter analysis. Values indicate percentage of positive cells. before electrophoresis or enzyme treatment. 6 Reactivity was determined by fluorescent analysis and cell sorting. c ND, not determined. Sodium dodecyl sulfate:polyacrylamide electrophoresis was carried

OCTOBER 1983 4813

Table 2 Zipf ef a/. (25) have described 2 monoclonal antibodies (1E7 Reactivity of monoclonal antibodies against human leukemic cell lines and 17F12) that react with the normal T-lymphocytes. The CellHL-60ML3KG1K562(S)Nairn reactivity patterns of S3.13 and S5.7 partially overlap with those (465)699 (137)2 leukemiaPromyelocytic of 1E7 and 17F12, since all 4 antibodies react with thymocytes leukemiaMyeloblastic (612)99 (133)10 and peripheral blood T-cells. However, S3.13 and S5.7 also react leukemiaErythroleukemiaNon-T-, (474)99 (110)14 (623)99 (166)92 with a subset of monocytes. 1DaudiRajiJurkatHUTleukemiaB-cellnon-B-cell (488)99 (207)1 Antibody OKT10, like S5.7 and S3.13, reacts with a discrete lymphomaB-cell (752)99 (123)1 lymphomaT-cell (818)99 (331)97 number of immature bone marrow cells (myeloid and lymphoid). leukemiaT-cell (409)99 (654)50 However, OKT10 does not react with normal mature T-cells or leukemiaHistiocytic 78U937THP1OriginPromyelocytic (899)10 (173)53 monocytes (9). leukemiaMonocytic (229)70 (166)7 leukemiaS5.796a (630)S3.131a (420) Antibody TA-1 is reactive with all T-lymphocytes and mono Percentage of positive cells determined by flow cytofluorimetry and confirmed cytes as well as with 13% of nucleated marrow cells (15). by radioimmunoassay as described in "Materials and Methods." However, TA-1 is nonreactive with cells of the eosinophilic or B- 6 Numbers in parentheses, mean intensity of fluorescence calculated from the cell lineage. The reactivity of S3.13 appears to overlap, in many relative intensity in 1000 channels. respects, the reactivity of monoclonal antibody RFB-1 described by Bodger ef a/. (2). Both antibodies react with a large fraction promyelocytic leukemia cell lines. S5.7 reacted with T-, B-, null, of peripheral T-cells and myeloblasts but react poorly with pro- and myeloid cell lines but reacted very weakly with U937 histio- myelocytes and a fraction of thymocytes. Both antibodies also cytic leukemic cells. S3.13 and S5.7 did not react with human bind to colony-forming cells (5) but do not react with pre-B-blasts fibroblasts, melanoma, colon carcinoma, teratocarcinoma, hep- or B-lymphocytes. Unlike RFB-1, however, S3.13 reacts weakly atoma, gastric carcinoma, or astrocytoma cell lines, but both with a fraction of monocytes. reacted with 4 neuroblastoma cell lines (data not shown). The 2 antibodies reported here define antigens that are abun Monoclonal Antibodies S5.7 and S3.13 Recognize Different dantly expressed in human leukemic cell lines, as well as on Target Structures. As shown in Fig. 1, monoclonal antibody acute leukemia cells freshly obtained from patients (21). These S5.7 immunoprecipitates a target protein of M, 20,000 which antigens are also present in hemopoietic progenitor cells of can be labeled by either cell surface radioiodination (Fig. 1, Lane various lineages4 (5), showing a complex pattern of distribution C) or metabolically by i_-[35S]methionine (Fig. 1, Lane G). The T- during the process of terminal differentiation. Work is in progress cell-specific glycoprotein T3, detected by monoclonal antibody to define the functions of such molecules, since this information Leu-4 (Fig. 1, Lanes B and F), has a similar molecular weight. In would be useful in understanding the significance of appearance contrast, S3.13 precipitates a protein of M, 29,000 when the and disappearance of the antigens at a defined differentiation cells are metabolically labeled with L-[35S]methionine (Fig. 1, Lane stage in each cell lineage. H). In several attempts, we were unable to detect the target antigen for S3.13 after cell surface radioiodination (Fig. 1, Lane ACKNOWLEDGMENTS D). The proteins immunoprecipitated, respectively, could also be immunoprecipitated by S5.7, and by S3.13, they could also be We thank Elaine Burton for typing and Marina Hoffman for editing the manu script. We thank Dr. Schtessinger, Dr. Andrews, and Dr. Aden for the gift of the immunoprecipitated from T-cell lines, AML cells, and normal T- neuroblastoma and teratocarcinoma cell lines. cells. REFERENCES

DISCUSSION 1. Billing, R. J., Rafizadeh, B., Drew, I., Hartman, G., Gale, R., and Terasaki, P. Human B lymphocyte antigens expressed by lymphocytic and myelocytic We have characterized the reactivity of 2 monoclonal antibod leukemia cells. I. Detection by rabbit antisera. J. Exp. Med., 144: 167-178, 1976. ies (S3.13 and S5.7) with a variety of human leukemic cells as 2. Bodger, M. P., Francis, G. E., Delia, D., Granger, S. M., and Janossy, G. A well as with normal hemopoietic cells. The same molecules (M, monoclonal antibody specific for immature human hemopoietic cells and T 29,000 and 20,000), which are precipitated from the HPB T-cell lineage cells. J. Immunol., 727: 2269-2274,1981. 3. Borst, J., Prendiville, M. A., and Terhorst, C. Complexity of the human T line and from the other T-lines, have been also precipitated from lymphocyte-specific cell surface antigen T3. J. Immunol., 128: 1560-1565, normal T-cells and AML cell lines (data not shown). Therefore, 1982. 4. Boyum, A. Separation of leukocytes from blood and bone marrow. Scand. J. the antigens represent polypeptides that are expressed early Clin. Lab. Invest., 21 (Suppl. 97): 7, 1968. during the process of hemopoietic differentiation and remain 5. FerrerÃ², D., Broxmeyer, H. E., Pagliardi, G. L, Venuta, S., Lange, B., Pessano, S., and Rovera, G. Antigenically distinct subpopulations of myeloid progenitor expressed in a few cell subsets during maturation. Monoclonal cells (CFU-GM) present in human peripheral blood and marrow. Proc. Nati. antibodies reactive with M, 20,000 to 30,000 polypeptides have Acad. Sei. U. S. A., 80: 4114-4118, 1983. been described by Hercend ef al. (6) and by Kersey ef al. (11). 6. Hercend, T., Nadler, L. M.. Pesando, J. M., Reinherz, E. L., Schlossman, S. F., and Ritz, J. Expression of a 26,000-dalton glycoprotein on activated human Antibody J2 recognizes a M, 26,000 glycoprotein present in T cells. Cell. Immunol., 64:192-199, 1981. activated T-cells but absent in resting T-cells (6). J2 also reacts 7. Hoffman, R. A., Kung, P. C., Hansen, W. P., and Goldstein, G. Simple and with platelets, but not with monocytes, and is therefore clearly rapid measurement of human T lymphocytes and their subclones in peripheral blood. Proc. Nati. Acad. Sei. U. S. A., 77: 4914-4917,1980. distinct from antibodies S3.13 or S5.7. 8. Janossy, G., Goldstone, A. J., Capellaro, D., Greaves, M. F., Kulen Kampff, J., Antibody BA2 immunoprecipitates a polypeptide of M, 24,000 Pippard, M., and Welsh, D. Differentiation linked expression of p28,33 (la-like) (11). This peptide is detectable on the surface of normal bone structures on human leukemic cells. Br. J. Haematol., 37: 391-402,1977. 9. Janossy, G., Tidman, N., Papageorgiou, E. S., Kung, P. C., and Goldstein, G. marrow lymphopoietic precursors in 77% of non-T- and non-B- Distribution of T lymphocyte subsets in the human bone marrow and thymus, ALL and 18% of T-ALL, but it is not present on peripheral blood leukocytes. ' D. FerrerÃ², S. Pessano, and G. Rovera, unpublished data.

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an analysis with monoclonal antibodies. J. Immunol., 726: 1608-1613,1981. Biochem. J., 724: 921-927, 1971. 10. Kearney, J. F., Radbruch, A., Liesegang, B., and Rajewski, K. New mouse 19. Minowada, J., Sagawa, K., Lok, M. S., Kubonishi, I., Nakazawa, S., Tatsumi, myeloma cell line that has lost immunoglobulin expression but permits the E., Ohnuma, T., and Goldblum, N. In: D. Serrou and C. Rosenfeld (eds.), construction of antibody secreting hybrid cell lines. J. Immunol., 723: 1548- International Symposium on New Trends on Human Immunology and Cancer 1550,1979. Immunotherapy, pp. 188-199. Paris: Doin, 1980. 11. Kersey, J. H., LeBien, T. W., Abramson, C. S., Newman, R., Sutherland, R., 20. Perussia, B., Trinchieri, G., Lebman, D., Jankiewicz, J., Lange, B., and Rovera, and Greaves, M. A human leukemia-associated and lymphohemopoietic pro G. Monoclonal antibodies that detect differentiation surface antigens on human genitor cell surface structure identified with monoclonal antibody. J. Exp. Med., myelomonocytic cells. Blood, 59: 382-392, 1982. 753:726-731,1981. 21. Pessano, S., FerrerÃ², D., Palumbo, A., Bottero, L., Hubbell, H., Lange, B., and 12. Knapp, W. (ed.). Leukemia Markers. New York: Academic Press, Inc., 1981. Rovera, G. Differentiation antigens of normal and leukemic myelomonocytic 13. Kohler, G., and Milstein, C. Continuous culture of fused cells secreting anti cells. In: D. W. Golde and P. A. Marks (eds.), Normal and Neoplastic Hemo- bodies of predefined specificity. Nature (Lond.), 246: 495-497,1975. topoiesis, UCLA Symposia on Molecular and Cellular Biology, Vol 9. New 14. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head York: Alan R. Liss, Inc., in press, 1983. of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970. 22. Reinherz, E. L., and Schlossman, S. F. The differentiation and function of 15. LeBien, T. W., and Kersey, J. H. A monoclonal antibody (TW-1) reacting with human T lymphocytes. Cell, 79: 821-827, 1980. human T lymphocytes and monocytes. J. Immunol., 725: 2208-2214,1980. 23. Schlossman, S. F., Chess, L., Humphreys, R. E., and Strominger, J. L. 16. Lebman, D., Trucco, M., Bottero, L., Lange, B., Pessano, S., and Povera, G. Distribution of la like molecules on the surface of normal and leukemic human A monoclonal antibody that detects expression of transferrin receptor in human cells. Proc. Nati. Acad. Sei. U. S. A., 73: 1288-1292, 1976. erythroid precursor cells. Blood, 59: 671-678,1982. 24. van Agthoven, A., Terhorst, C., Reinharz, E., and Schlossman, S. Characteri 17. LeClair, K. A., van Agthoven, A., and Terhorst, C. A rapid method for isolation zation of T cell surface glycoproteins T1 and T3 present on all human peripheral of antigenically active human cell surface antigens associated with fe-micro- T lymphocytes and functionally mature thymocytes. Eur. J. Immunol., 77: globulin using a monoclonal antibody. J. Immunol. Methods, 41: 137-144, 18-21,1981. 1981. 25. Zipf, T. F., Fox, R. I., Dilley, J., and Levy, R. Definition of the high risk acute 18. Marchalonis, J. J., One, R. E., and Samter, J. V. Enzymatic iodinationâ€”probe lymphoblastic leukemia patient by immunological phenotyping with monoclonal for accessible surface proteins of normal and neoplastic lymphocytes. antibodies. Cancer Res., 47: 4786-4789, 1981.

ABCD EFGH MW x10

Fig. 1. Analysis of the radiolabeled target antigens of S5.7 and S3.13 by sodium dodecyl sulfate:polyacrylamide gel electrophoresis and autoradiography. HPB ALL cells were radiolabeled with Na125l catalyzed by lactoperoxidase and hydrogen peroxide (Lanes A to D) or by culturing in the presence of u-^Slmethionine (Lanes Â£to F). Immunoprecipitation was done as described in 'Materials and Methods." The antigens were analyzed by sodium dodecyl sulfate:polyacrylamide gradient gels (10 to 15% in acrylamide) under reducing conditions according to the method of Laemmli (14). Molecular weight markers were cytochrome c (M, 13,000), carbonic anhydrase (M, 29,000), ovalbumin (M, 45,000), bovine serum albumin (M, 69,000), and phosphorylase B (M, 94,000). Lane A, 125l-labeled HPB ALL cells, control ascites; Lane B, 12Sl-labeled HPB ALL cells, Leu-4; Lane C, 125l-labeled HPB ALL cells, S5.7; Lane D, 125l-labeled HPB ALL cells, S3.13; Lane E, ^S-labeled HPB All cells, control ascites; Lane F. ^S-labeled HPB ALL cells, Leu-4; Lane G, ^S-labeled HPB ALL cells, S5.7; and Lane H, ^S-labeled HPB ALL cells, S3.13.

OCTOBER 1983 4815

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1983 American Association for Cancer Research. Differentiation Antigens of Human Hemopoietic Cells: Patterns of Reactivity of Two Monoclonal Antibodies

Silvana Pessano, Lisabianca Bottero, Jeffrey Faust, et al.

Cancer Res 1983;43:4812-4815.

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