[CANCER RESEARCH 48, 5759-5765, October 15, 1988] New Hematopoietic Differentiation Antigens Detected by Anti-K562 Monoclonal Antibodies1 FrançoiseFarace,2 Marie-Thérèse Mitjavila,3 AH Betaieb, Marie Christine Dokhelar, JoëlleWiels, Yvonne Finale, Nelly Kieffer, Jeanine Breton-Gorius, William Vainchenker, and Thomas Tursz Groupe d'Immunobiologie des Tumeurs U.A. 1156 CNRS, Institut Gustave Roussy, Rue Camille Desmoulins, 94SOS Villejuif, France [F. F., M. C. D., J. W., Y. F., T. T.], and INSERM U 91, Hôpital Henri Mondar, Creteil, France [M-T. M., A. B., N. K., J. B-G., W. V.J ABSTRACT Following immunizations of BALB/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immu- noprecipitates a M, 105,000 glycoprotein on K562 cells, (b) Two MoAbs, 14B6 and 12111.react with cells of the monocytic series. MoAb 14B6, which also faintly stains platelets, is reactive with immature myeloid cells and the majority of hematopoietic progenitors. The 14B6 antigen has been immunoprecipitated from 12-0-tetradecanoylphorbol-13-acetate treated K562 cells as a M, 130,000-100,000 protein. Antigen 12B1 is expressed only on cultured monocyte/macrophages and is restricted to a subpopulation of monocytes and to follicular dendritic cells. It is not detected on hematopoietic progenitors. Immunoprecipitation experiments performed on 12-0-tetradecanoylphorbol-13-acetate treated K562 cells revealed a glycoprotein with a molecular weight of 93,000-86,000. (<•) Two anti-K562 MoAbs, CF4 and I IK1(1.recognize a myeloid differentia tion antigen expressed from the granulomonocytic colony forming unit stage to polymorphonuclear neutrophils. These MoAbs detect an appar ently original glycolipid moiety distinct from LeX. (d) Two MoAbs recognize antigens expressed on the granulomonocytic series. 2E1 rec ognizes the monocyte low affinity Fc receptor (M, 40,000) and defines a new cluster of myeloid differentiation (CDw32). The antigen is expressed on a small portion of immature hematopoietic progenitors. 8F5 identifies aM 95,000 protein which is also present on plasma cells. In some experiments, it is detected on erythroid colony forming unit analysis. Immunizations with K562 cells thus resulted in the production of anti bodies recognizing antigens of the monocytic, granulocytic, as well as erythroid series. However, three of them are also detected on hemato poietic progenitors. INTRODUCTION The K562 cell line (1) was considered originally as a granu locytic cell line on the basis of (a) positive staining with human antisera directed against granulocytic antigens and (b) the ab sence of lymphoid markers (2, 3). The presence of hapten X (CD 15), an antigen which is mostly associated with granulocytic differentiation, has been demonstrated on the surface of K562 cells (4, 5). However, it was shown later that K562 cells exhibit markers of the erythroid series, including hemoglobin and glycophorin A (6-8). Exposure to hemin or sodium butyrate favors hemoglobin synthesis (9-11) but does not induce termi nal erythroid differentiation. A peroxidase activity cytochemi- cally identical to that of platelet peroxidase has been detected Received 2/16/88; revised 6/10/88; accepted 7/19/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'This work was supported by Grants from INSERM (Nos. 842018 and 843003) (Institut National de la Santé et de la Recherche Médicale, Paris, France), from Institut Gustave Roussy, Villejuif. and from Association pour la Recherche sur le Cancer, Villejuif. 2To whom requests for reprints should be addressed. in rare cells of the K562 cell line (12). After TPA3 or sodium butyrate induction, the number of cells exhibiting this peroxi dase activity increased with concomitant appearance of platelet glycoprotein Ilia (13, 14). Therefore, whether K562 is a pure erythroleukemic cell line simultaneously expressing early and late markers of erythroid differentiation or an abnormal pluri- potent cell line is still a matter of debate. The purpose of this study was to analyze a panel of mono clonal antibodies obtained after immunization with K562 cells, assuming that characterization of the antibodies would provide further elements concerning cell lineage specific antigens pres ent on these cells. We show here that immunization with KS62 cells resulted in the production of antibodies recognizing new antigens specific for the monocytic series as well as other original antibodies directed against granulocytic and erythroid antigens. MATERIALS AND METHODS Isolation of Monoclonal Antibodies Female BALB/c mice, 4 months old, were given injections of K562 cells (IO7 cells by injection). Three days after the last immunization, the spleen was removed and gently teased. Splenocytes were fused with the mouse myeloma cell line NS1 according to the technique of Köhler and Milstein (15). Briefly, spleen cells from the immunized mice were added to myeloma cells in a ratio of 4 to 8 lymphocytes per myeloma cell and fused in the presence of 50% polyethylene glycol. Cells were then grown in microplates (Falcon, Oxnard, CA) containing RPMI 1640 (Gioco, Grant Island, NY), supplemented with hypoxanthine- aminopterin-thymidine, 15% heat inactivated horse serum (Gibco), i glutamine (2 m\i). and gentamicin (50 mg/liter). Supernatants from wells containing growing hybrids were screened by ELISA assay on K562 cells. Growing hybridomas secreting specific antibodies were subcloned by the limiting dilution technique. Control Monoclonal Antibodies 80H5 (anti-LeX, CD15) reacts with granulocytes, their precursors, and monoblasts ( 16); C17 is an anti-platelet GP Ilia MoAb ( 17); LICR/ LON/R10 is an anti-glycophorin A MoAb (18); and OKM1 is an anti- C3bi receptor MoAb (CDU), which stains monocytes, granulocytes, and null cells (19). Cell Lines K562, HL60 (20), and U937 (21) cells were routinely cultured in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, L-glutamine (2 mM), and gentamicin (50 mg/liter). Differentiation Induction with Hemin, TPA, and DMSO. K562 cells were treated with 0.1 mM hemin (type III; Sigma) for 5 days. TPA (Sigma, St. Louis, MO) induction of K562, HL60, and U937 cells was performed by treating the cells with 160 nM TPA for 48 h. DMSO 3 Supported by a Catalan Institution (CIRIT). 4 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; MoAb, monoclonal antibody; HI 1 I . erythroid burst forming unit; CFU-E, erythroid colony forming unit; CFU-GM, granulomonocytic colony forming unit; CFU-MK, megakaryocytic colony forming unit; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; ELISA, enzyme linked immunosorbent assay. 5759 on July 16, 2015. © 1988 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from