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.     

Canine lymphoma-associated antigens defined by murine monoclonal antibodies

Summary Lymphoma in dogs resembles human non-Hodgkin's lymphoma in pathological presentation, immunophenotype, and response to therapy, thus representing a good model for comparative studies with human disease. Monoclonal antibodies (MAbs) were derived from mice immunized with a dog lymphoma cell line. Three MAbs were selected for further application in immunophenotyping and immunotherapy. The binding specificities, antigen characterization, and isotypes for these MAbs are described.Supported by NCI grant CA-10815     

Two surface antigens expressed on proliferating mouse T lymphocytes defined by rat monoclonal antibodies

A hybrid cell line resulting from the fusion of a Con A-activated normal mouse spleen cell and a transformed mouse T cell (EL-4BU) has been used to prepare and select rat monoclonal antibodies reactive with molecules expressed on the surface of proliferating, as opposed to resting, mouse T cells. In this report, the characterization of two such antigens identified in this way is described. One antigen is a membrane component common to mitogen-activated T and B cells, some bone marrow cells, and various transformed cell lines but is not detectable on either normal thymocytes or the majority of spleen cells by radioimmunoassay or FACS analysis. It has a m.w. of approximately 200,000 daltons under nonreducing conditions and 100,000 daltons under reducing conditions. Antibodies to this antigen precipitate cell-bound transferrin but do not react directly with transferrin itself. It would thus appear that the antigen is the transferrin receptor molecule. The second antigen is not detectable on normal thymocytes, spleen cells, bone marrow cells, or mitogen-stimulated spleen cells but is expressed at high levels on some transformed T cell lines. It, too, appears to be a dimer, with a m.w. of 95,000 daltons under nonreducing conditions, decreasing to 50,000 daltons under reducing conditions. Although the function of the 95,000-dalton antigen is not yet known, its lack of expression on adult T cell populations both before and after activation suggests either a short-lived role at a very early stage of T cell development and/or an association with T cell transformation.     

Phylogenic distribution of human renal antigens defined by monoclonal antibodies

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.     

Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies

Monoclonal antibodies directed against human cancer cells were prepared by the murine hybridoma technique. These antibodies detect Lewis blood group antigens as determined by indirect solid-phase radioimmunoassay, hapten inhibition studies, and chromatogram binding assay. One monoclonal antibody is specific for the Lea terminal carbohydrate of Gal beta 1----3Glc NAc(4----1 alpha Fuc) beta 1----3LacCer. Five monoclonal antibodies react with the Leb terminal carbohydrate sequence of Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer, and four of these antibodies are highly specific for this glycolipid and do not react with other similar di- and monofucosylated glycolipids. One of the anti-Leb antibodies cross-reacts with blood group H glycolipid and has binding properties similar to those of the previously described antibody NS-10-17 [M. Brockhaus, J. L. Magnani, M. Blaszczyk, Z. Steplewski, H. Koprowski, K.-A. Karlsson, G. Larson, and V. Ginsburg (1981) J. Biol. Chem. 256, 13223-13225]. Two antibodies react with both the Lea and Leb antigens, though both bind preferentially to Leb.     

Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies

Three differentiation antigens of mouse teratocarcinoma stem cells are defined using a panel of ten IgM-class monoclonal antibodies raised against teratocarcinoma F9 cells. TEC-01 and four other antibodies define an antigen that corresponds to SSEA-1. TEC-02 antibody defines an antigen that is expressed on teratocarcinoma stem cells, parietal yolk sac cells PYS-2, unfertilized eggs including the zona pellucida and blastocysts. It is absent from all mouse adult tissues tested. Three other antibodies exhibit binding properties similar to TEC-02. TEC-03 antibody defines an antigen that is expressed on teratocarcinoma stem cells, PYS-2 cells and mouse blastocysts. It is absent from all mouse adult tissues except for lungs.     

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.     

Heterogeneity of human melanoma-associated antigens defined by monoclonal antibodies and conventional xenoantisera

Summary Immunochemical analysis of cultured human melanoma cell detergent extracts and spent culture medium with conventional xenoantisera and monoclonal antibodies identified four types of 94,000 (94K) dalton molecules and two types of high-molecular-weight melanoma-associated antigens by the following characteristics: (1) association with other components, (2) mobility in SDS-PAGE under reducing and nonreducing conditions, (3) antigenicity, and (4) presence in spent culture medium. Conventional xenoantisera were found to contain antibody populations to antigenically distinct structures, some of which have similar apparent molecular weights. Immunodepletion studies showed that the antigenic determinant detected by the monoclonal antibody 225.28S to a high-molecular-weight melanoma-associated antigen was expressed on a subpopulation of the antigens defined by the conventional xenoantiserum #8995. These data prove that antibodies reactive with antigens of similar molecular weight cannot be assumed to identify the same structures, and indicate that tumor-associated antigens may be heterogeneous in the expression of antigenic determinants defined by monoclonal antibodies.Visiting investigator from the Veterans Administration Hospital, Minneapolis, MinnesotaVisiting investigator from Sapporo Medical College (Japan). Abbreviations used: MAA, melanoma-associated antigen; PBS, phosphate-buffered saline; NP40, nonidet P40; MoAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 2-ME, 2-mercaptoethanol     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

Epitope clusters of Qa-2 antigens defined by a panel of new monoclonal antibodies

A recently derived intra-MHC recombinant mouse strain, the C3H.KBR was found to produce a surprisingly high titer of anti-Qa antibodies when immunized with C3H.SW lymphocytes. By using this immunization combination, a panel of 10 mAb with specificity for determinants encoded by the Q region was produced. These reagents were analyzed for strain distribution by microcytotoxicity, immunofluorescence, and flow cytometry assays. Competitive inhibition analyses, performed by using fluorescein-labeled antibodies and normal spleen cells, defined at least three epitope clusters, or groups of spatially related determinants, detected by this panel. One epitope cluster was unique to this new series of antibodies in that it was not detected with seven previously described anti-Qa mAb. These antibodies also have been analyzed for reactivity with products of isolated Q-region genes by using transfected cell lines. The data indicate that the Q6d, Q7d, and Q10d genes encode determinants reactive with one or more mAb and that two of the three epitope clusters defined with normal cells map to the N and/or C2 domains of these molecules. The third epitope cluster is presumed to map to the C2 domain. These reagents should be useful in determining the number of Q-region genes expressed and in analyses of Q gene expression in subpopulations of normal cells, in transfected cell lines, and during differentiation and ontogeny.     

Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies

Endothelial cells play an essential role in immune responses by regulating the entry of leukocytes into lymphoid tissues and sites of inflammation. As an initial approach to analyzing endothelial cell specialization in relation to such immune function, we have produced monoclonal antibodies (MAB) against mouse lymph node endothelium. Three antibodies were selected: MECA-20, recognizing the endothelium of all blood vessels in lymphoid as well as non-lymphoid organs; MECA-217, which stains the endothelium lining large elastic arteries, but among small vessels is specific for post-capillary venules within lymphoid organs and tissues exposed to exogenous antigen, such as skin and uterus; and MECA-325, an antibody that demonstrates specificity for the specialized high endothelial venules (HEV) that control lymphocyte homing into lymph nodes and Peyer's patches. MECA-325 failed to stain vessels in any non-lymphoid organs tested. Immunoperoxidase studies of HEV in lymph node frozen sections, and of isolated high endothelial cells in suspensions, demonstrated that the antigens recognized by all three antibodies are expressed at the cell surface; those defined by MECA-20 and MECA-325 are also present in the cytoplasm. To study the regulation of the antigens defined by these MAB in relation to extra-lymphoid immune reactions, we assessed their expression in induced s.c. granulomas as a model for chronic inflammation. Small vessels in the granulomas were already stained by MECA-217 in the first days of development. In contrast MECA-325 detected postcapillary venules (which frequently displayed the morphologic characteristics of HEV) only from approximately 1 wk, in parallel with the development of a persistent mononuclear cell infiltrate including numerous lymphocytes. The selective appearance of the MECA-325 antigen on vascular endothelium supporting lymphocyte traffic in both lymphoid and extra-lymphoid sites suggests that this antigen may play an important role in the process of lymphocyte extravasation. The demonstration of lymphoid organ- and inflammation-specific microvascular antigens offers direct evidence for a complex specialization of endothelium in relation to immune stimuli, and supports the concept that microvascular differentiation may play an important role in local immune responses.     

Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies

A series of seven monoclonal antibodies directed at determinants on human peripheral blood monocytes were produced and characterized. The antibodies were separated into three groups based on cell distribution and percentages of monocytes bearing antigen. Hybridoma antibodies, termed OKM1, OKM9, and OKM10, recognized antigen(s) expressed on the majority of adherent monocytes, null cells, and granulocytes. The second group, comprising OKM5 and OKM8, reacted with most adherent monocytes and platelets. OKM3 and OKM6, comprising a third group of antibodies, reacted with a subpopulation of adherent monocytes and platelets. OKM antibodies were not expressed on lymphocytes, thymocytes, and lymphoblastoid cells, with the exception of OKM3 which reacted with three B-cell lines. SDS gels of immunoprecipitates formed with OKM antibodies yielded the following tentative molecular weight results: OKM1 and OKM9 antigens appeared to be 160,000 (nonreduced) and 170,000 (reduced); OKM10 precipitated two polypeptides of 170,000 and 115,000 (reduced); OKM5 and OKM8 precipitated a single polypeptide of 88,000 (reduced, nonreduced); OKM6 antigen appeared to be 116,000 (nonreduced) and 130,000 (reduced).     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.