Expression of a 21KD molecule on hematopoietic progenitor cells.

A monoclonal antibody (MoAb) K15 (IgG3) was obtained by fusion between SP2/0, mouse myeloma cell line and spleen cells from BC3Fl mice immunized with K562. a chronic myelocytic leukemia (CML) blastic crisis cell line. MoAb Kl5 precipitated a 21 kilodalton (KD) polypeptide in reduced condition. It stained lymphocytes, monocytes, eosinophiles and small bone marrow cells, but not neutrophiles, platelets, red blood cells (REC) and large bone marrow cells. Depletion of K15 + cells abolished both colony-forming units-granulocyte/monocyte (CFU-GM) and burst-forming units-erythroid (BFU-E). Thus MoAb Kl5 identifies antigens expressed on hematopoietic progenitor cells. The selective retention of this antigen by certain cell lineages may suggest some functional role of this antigen. Key wo1·ds: Monoclonal antibody, CFU-GM, BFU-E Monoclonal antibodies (MoAb) against immature hematopoietic cells have been describedl. We pay attention to the phenotype of human bone marrow progenitor cells. We are attempting to produce antiprogenitor cell reagents that can identify comitted progenitor cells of CFU-GM and BFU-E. This kind of reagents is useful for analysis of bone marrow progenitor cells. Utilizing non-lymphoid cell lines as immunogens, we have established a series of monoclonal antibodies and shown to have selective reactivity with non-lymphoid cells. One of them (Kl5) have been studied more extensively. MoAb K15 have the reactivity with progenitor cells of CFU-GM and BFU-E as well as with more mature hematopoietic cells. This report describes our investigation with MoAb Kl5. MATERIALS AND METHODS Generation of MoAb K562, CML blastic crisis cell line was used as an immunogen. Ten-week-old BC3Fl female mice were injected i.p. with 2 x 10 cells with 4mg alum as adjuvant. A second immunization was done 3 weeks later with 2 x 10 cells in phosphate buffered saline (PBS) i.p .. Three days later, spleen cells were fused with SP2/0 Ag 14 tumor cells with PEG 1000. Hybridomas were selected in HAT medium: their supernatant were screened by indirect immunofluorescence. The desired hybridomas were cloned on agarose. Details of these procedures have been describedl. Cell preparation Defibrinated blood from normal volunteers was used as a source of peripheral blood mononuclear cells (PBMC). PBMC were separated as describedl. Monocytes were separated by Percoll continuous gradient centrifugationl. Granulocytes were separated by Percoll discontinuous gradientl. The purity of the granulocyte fraction was more than 97% as determined by Wright-Giemsa staining. Platelets were isolated from platelet-rich plasma and red blood cell (REC) from the pellets of Ficoll-Hypaque gradients of peripheral blood cells. Identification of antigen K15 bearing granulocytes determined by immune rosette method. Separated granulocytes were incubated with hybridoma culture supernatant, washed three times with PBS and mixed with ox-REC conjugated with anti-mouse Ig by CrC13 method l. After standing for an hour, the cells were smeared on slide glasses and stained with Wright-Giemsa solution. The type of rosette forming cells was determined under the microscope. Bone marrow cells and bone marrow culture Bone marrow cells were obtained from normal volunteers. They were separated by Ficoll-Hypaque density centrifugation to remove REC and mature granulocytes. Separated bone marrow cells were subjected to an immune rosette methodl to obtain cells reactive with MoAb K15, or they were subjected to a complement (C)-mediated cytolysis procedure to deplete the reactive cells. For the Cmediated killing procedure, 0.5 ml of 2 x 10/ml of bone marrow cells incubated with 0.5 ml of hybridoma culture supernatant for 30 min at 37°C. One milliliter of baby C (Pel Freez Biologicals, Rogers, AR, U.S.A.) was added and the mixture was incubated for 45 min at room temperature. 110 M. Takaishi and S. M. Fu Dead cells were removed by centrifugation on a Ficoll-Hypaque gradient. Bone marrow cells were cultured in quadruplicates in Iscove's modified Dulbecco's medium containing 0.9% methylcellulose, 10% conditioned medium from human peripheral blood leukocytes stimulated with phytohemagglutinin, and 30% fetal bovine serum (FBS). This procedure was essentially as described by Messner et all BFU-E were scored as hemoglobin-containing single or multiple colonies of more than 64 cells on the 14th day. This culture also allowed CFU-GM colony formation. CFU-GM were scored as colonies of more than 40 cells on the 14th day, since the growth of CFU-GM was slow in our cultures. For the preparation of Esigbone marrow cells, separated bone marrow cells were mixed with neuraminidase-treated sheep RBCl and anti-human IgM goat antibody-conjugated ox-RBCl. After one hour incubation at 4 °C, cells were subjected to Ficoll-Hypaque density centrifugation to deplete the cells which formed rosettes with neuraminidasetreated sheep REC and anti-human IgM-conjugated ox-REC. Immunofluorescence studies Cells (0.05 to 1 x 10) were first incubated with hybridoma culture supernatants for 20 min at 4 °C. After three washings with PBS containing 1 % bovine plasma albumin, fluorescein-labeled F (ab')2 anti-mouse lg goat antibody (Tago, Burlingame, CA, U.S.A.) was added and a 20 min incubation at 4 °C was performed. After three washings, cells were analyzed with a Epics V flow cytometry (Coulter Electronics). Integrated fluorescence of the population gated by forward angle light scatter and right angle light scatter was measured and 10, 000 cells were analyzed. Iodination and immunoprecipitation Cells were iodinated in suspension by the method of Hubbard and Cohnl. In brief, 2 x 10 K562 cells were incubated with 1mCi/ml NaI, 50m U /ml type V glucose oxidase (Sigma Chemical Co., St. Louis, Mo, U.S.A.) and lOμg/ml lactoperoxidase (Calbiochem-Behring Co., San Diego, CA, U.S.A.) for 5 min on ice. The reaction was terminated by aspirating the supernatant and by repeated washings with RPMI 1640. After iodination, Immunoprecipitation, gel electrophoresis, and autoradiography were performed as describedl. RESULTS Characterization of MoAb K15 MoAb K15 was established with K562 cells as an immunogen. By ELISA, MoAb K15 was typed to be IgG3. MoAb K15 precipitated a molecule with 21 KD from 1-labeled K562 cells under reduced condition (Fig. 1, lane 1). Distribution of antigen K15 on peripheral blood cells The cellular distribution of the reactive antigens by MoAb K 15 was analyzed by immunofluoresMrx10-3