Expression of CD15 (FAL) on myeloid cells and chromosomal localization of the gene

Summary Different CD15 murine monoclonal antibodies were studied. These antibodies appeared to react specifically with the human myeloid-lineage-derived cell types in both peripheral blood and bone marrow.The antigens recognized by these antibodies were immunoprecipitated from lysates of¹²⁵I-labelled neutrophilic PMNs of healthy donors and subsequently analysed by electrophoresis on SDS-polyacrylamide gel and autoradiography. All antibodies precipitated the same membrane polypeptides from the membrane-iodinated PMN lysates: 105 and 150-kDa as most prominent, together with 260-, 230-, 67- and 52-kDa polypeptides.Absorption studies were performed with synthesized carbohydrate molecules. Antibody B4.3 appears to be directed against 3--fucosyl-N-acetyl-lactosamine (FAL). Competition experiments with¹²⁵I-labelled B4.3 demonstrated complete inhibition of binding by B4.3 and three other CD15 antibodies (VIM D5, UJ308, MI/N1), and partial inhibition by three additional antibodies (FMC10, FMC12, FMC13), indicating binding to the same antigenic structure. None of the antibodies reacted with monocytes using the immunofluorescence technique, but after neuraminidase digestion of these cells, positive reactions were obtained with all antibodies. Immunoprecipitation with lysates of both native and neuraminidase-digested monocytes showed no polypeptide bands. Monocytic differentiation of the myeloid cell line HL60 by 12-O-tetradecanoylphorbol-13-acetate (TPA) was accompanied by a decrease in reactivity with the antibodies, which could be reversed by neuraminidase digestion. This indicates that 3--fucosyl-N-acetyl-lactosamine is masked for the detection with antibodies upon monocytic differentiation by sialylation.Human x mouse myeloid cell hybrids were obtained after fusion of human myeloid cells and the HPRT-deficient murine myeloid cell line WEHI-TG. These hybrids were tested for reactivity with the anti-CD15 antibodies.The CD15 panel exhibited very similar reactivity patterns with the hybrid clones. Chromosomal analysis of hybrid cell metaphases revealed that the gene(s) involved in the expression of FAL must be located on human chromosome 11 in the region q12-qter.     

Suppression of the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells by hybridization with normal macrophages.

Mouse myeloid leukemic cells that multiplied in vitro and were malignant in vivo were hybridized with non-malignant non-multiplying normal mouse or human macrophages. Both the hybrids with mouse and with human macrophages were non-malignant and non-multiplying. The suppression of malignancy and cell multiplication in these hybrids was associated with the expression of eight other properties expressed in normal macrophages but not in the myeloid leukemic cells. These properties were C3 and Fc rosettes and immune phagocytosis, rosettes and phagocytosis of uncoated erythrocytes, synthesis and secretion of lysozyme and a high frequency of cap formation by concanavalin A (ConA). The hybrids also expressed two properties of the myeloid leukemic cells, a lack of cell attachment to the surface of a Petri dish and staining for myeloperoxidase. The use of specific antibodies has shown that the lysozyme produced by hybrids between human macrophages and mouse myeloid leukemic cells was human lysozyme. The results indicate that the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells was suppressed in hybrids with normal macrophages, and that his suppression can be dissociated from the normal macrophage property of cell attachment.     

Construction of human-mouse T cell hybrids that express human T cell-associated surface antigens and allow the chromosomal localization of these antigens

We have constructed somatic cell hybrids between the murine T cell line BW5147 and cells from patients suffering from T cell acute lymphoblastic leukemia. The obtained hybrid clones were analyzed for expression of human T cell antigens and presence of human chromosomes. T cell hybrids derived from fusion between the BW5147 cell line and bone marrow cells from a patient with pre-T acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1-/T6-/T4-/T8-/T3-) appeared to express the human T cell antigen Tp41, which can be recognized by the monoclonal antibodies 3A1 and WT1. Although this panel of hybrid cells contained all human chromosomes, no other T cell antigens were expressed. Fusion of the BW5147 cell line with peripheral blood cells from a patient with a more mature T cell acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1+/T6-/T4+/T8+/T3-) resulted in a panel of hybrid clones that expressed not only the Tp41 antigen, but also the human T cell antigens T1 and T4; two hybrids even expressed the T3 antigen. This panel of hybrids also contained the whole human genome. The two panels of human-mouse T cell hybrids allowed us to assign the genes coding for the human T cell antigens Tp41, T1, and T4 to human chromosomes 17, 11, and 12, respectively. Furthermore, these data support our previous suggestion that the expression of human lymphoid differentiation antigens in human-mouse lymphoid hybrids is influenced by the differentiation stage of the fusion partners.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.     

The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens

Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

A human hybrid myeloma for production of human monoclonal antibodies

We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid.     

Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.     

Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice.

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.     

Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.     

Expression of nonspecific cross-reacting antigen species in myeloid leukemic patients and healthy subjects

The reactivity of two monoclonal antibodies recognizing NCA-95 and NCA-55 (MAb 47 and MAb 192, respectively) with a polyclonal anti-NCA serum in myeloid leukemic cells isolated by density gradient centrifugation was compared using an immunofluorescence test (IF). It was observed that the blood myeloid cells in 78.8% of the patients with different types of myelocytic leukemias and all granulocytes of 15 normal donors showed similar expression of the NCA species studied. The leukocytes of the remaining patients did not synthesize the NCA-95 species regardless of the maturation stage of the cells studied. In two patients, synthesis of this NCA form was limited to the fractions containing myelocytes and metamyelocytes. We have found that all anti-NCA antibodies studied recognized different antigenic epitopes in a myeloid cell series. A relationship between the patient's survival and the proportion of NCA-containing cells was also observed.     

High frequency of natural autoantibodies in normal newborn mice

Spleen cells from 6-day-old nonimmunized BALB/c and BALB.B10 mice were fused with the nonsecreting hybridoma cell line Sp2/0. Three hundred and eighty-four immunoglobulin-secreting hybrids were screened for antibody activity against mouse actin, tubulin, and myosin, and against TNP, peroxidase, renin, DNA, and neurofilaments. At least 24 hybridomas in the collection (6.25%) exhibited antibody activity against this panel of antigens. Ten of these hybrids were cloned, were propagated, and the corresponding monoclonal IgM protein was isolated from ascitic fluids and was further characterized. At least four groups of antibody specificities were identified: 1) one clone reacting with TNP only; 2) one clone reacting with both actin and tubulin; 3) two clones which bound to both TNP and actin; and 4) a fourth group, comprising the six other clones, which all exhibited widespread reactivity and bound to actin, tubulin, myosin, and TNP. These results indicate: 1) B cell clones directed against self antigens are activated in the internal environment and are recovered consequently by somatic cell hybridization; 2) the widespread antibody specificities found for these newborn mouse antibodies are very similar to those previously characterized with human natural antibodies and human monoclonal Ig; and 3) the frequency of B cells binding to cytoskeletal proteins and TNP is very high (at least 6.25%).     

Properties of reticulum cell sarcomas in SJL/J mice. III. Promotion of tumor growth in irradiated mice by normal lymphoid cells.

Experiments were performed to elucidate the mechanisms by which lymphocytes obtained from an M-antigen-incompatible strain reduce the specific mixed lymphocyte culture (MLC) response of lymphoid cell populations after injection into allogeneic recipients. Mice of strain CBA were injected with spleen cells from hybrids of the H-2-compatible, M-antigen-incompatible strain C3H. Normal C3H × CBA spleen cells increased the MLC reactivity of the host's lymphocytes during the first 1–3 days, and thereafter the response against C3H was drastically reduced. Mitomycin-treated or antibody-coated C3H × CBA cells rather enhanced the MLC responsiveness. Roughly similar results were obtained by injecting untreated H-2-incompatible C3H hybrid lymphocytes. Lymph node or spleen cell populations from CBA mice, injected with C3H × CBA cells up to 2 weeks earlier, were found to depress the MLC reactivity against C3H when transferred to new CBA hosts. The results indicate that injected cells had survived for 2 weeks in the host. On the other hand, H-2-incompatible C3H hybrid cells could not be detected even at day 3 after injection into CBA mice. The results also indicate that C3H hybrid lymphocytes have to be functionally intact and able to survive in the host for a relatively long period of time to be able to reduce the specific MLC response of the host's lymphocytes.     

Antigenic immunostaining patterns in somatic hybrids of human HeLa cells and mouse fibroblasts 3T3.4E propagated in conventional medium and delipidized serum

Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (beta 2-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin). All of these antigens were expressed in HeLa cells cultured in conventional medium or with delipidized serum. Conversely mouse fibroblasts contained only vimentin, fibronectin and laminin. All the parental antigens were present in first passage hybrid cells cultured in conventional medium. Vimentin, fibronectin and laminin were maintained in fourth passage hybrids whereas keratins, beta 2-microglobulin, EGF and transferrin receptors were no longer detected. When propagated in medium with delipidized serum, hybrid cells re-expressed these antigens after 5 days of culture. These findings suggest that the reexpression of HeLa cell antigens in hybrid cells was related to deficiency in vitamin A.     

A GDP-fucose:[Gal beta 1----4]GlcNAc alpha 1----3-fucosyltransferase activity is correlated with the presence of human chromosome 11 and the expression of the Lex, Ley, and sialyl-Lex antigens in human-mouse cell hybrids

By fusion of human leukocytes and cells of the murine myeloid cell line WEHI-TG, we produced human-mouse myeloid cell hybrids. Hybrids which contain human chromosome 11 have been demonstrated to express the myeloid-associated carbohydrate antigen Lex (Geurts van Kessel, A. H. M., Tetteroo, P. A. T., Von dem Borne, A. E. G. Kr., Hagemeijer, A., and Bootsma, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3748-3752). In this paper, we report that the hybrids that contain chromosome 11 also expressed the Lex-related antigens Ley and sialyl-Lex. Glycosyltransferase activities were measured in a panel of six such hybrid cell lines, and the correlation to antigen expression and to the presence of human chromosomes was investigated. GDP-fucose:[Gal beta 1----4]GlcNAc alpha 1----3-fucosyltransferase activity in the hybrids tested correlated with the expression of Lex, Ley, and sialyl-Lex and with the occurrence of chromosome 11. No such correlation was found for several other glycosyltransferases involved in the synthesis of these antigens. These findings suggest that the gene for alpha 3-fucosyltransferase is located on chromosome 11 and that it is through the activity of this enzyme that the expression of Lex, Ley, and sialyl-Lex in human myeloid cells is regulated.