Monoclonal antibodies as neural cell surface markers

A comparison is made of the immunohistochemistry at the ultrastructural level of three monoclonal antibodies directed against surface components of CNS cells. Hybridomas secreting these antibodies were obtained from two cell fusions of a rat myeloma cell line and immune splenocytes derived from rats immunized either with primary mouse brain cultured cells or membrane components. In cultures one antibody, anti-BSP-2 (Brain Surface Protein-2), was preferentially directed against neurones while another, anti-BSP-3 (Brain Surface Protein-3), preferentially labeled astrocytes. In mouse cerebellar sections, both labeled the surface of Purkinje cells, granule cells and astrocytes. In addition a cytoplasm localization was apparent in granule cells and astrocytes. Another antibody anti-MESA-1 (Mouse Endothelial Surface Antigen-1) reacted exclusively with the surface of endothelial cells lining blood vessels. These data are discussed with reference to the biochemical nature of the corresponding antigens and to known glycoproteins of neural cell membranes.     

Isolation of subpopulations of murine epidermal cells using monoclonal antibodies against differentiation-related cell surface molecules

Abstract. Monoclonal antibodies (mAbs) against epithelial cells were prepared by immunization of rats with lyophilized murine epithelia. Screening against tissue sections and epithelial cell suspensions permitted identification of mAbs against surface molecules that are expressed early in cell differentiation. Staining with These mAbs followed by fluorescence-activated cell sorting enabled isolation of subpopulations of basal epithelial cells. Staining these subpopulations with antibodies against known differentiation markers (cytokeratins and bullous pemphigoid antigen) and measurements of cell size indicated that they represented fractions of the basal cell population in sequential stages of early differentiation. Labeling mice with bromodeoxyuridine at various limes prior to cell isolation showed that the least-differentiated basal cells cycle more slowly than those at later stages, data which support the concept of a differentiation-related, hierarchical pattern of organization of the proliferative compartment.     

Monoclonal antibodies specific for cell culture mycoplasmas

Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody to Mycoplasma pneumoniae was produced. M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis of M. pneumoniae infection in man.     

Monoclonal antibodies specific for cell culture mycoplasmas

Summary Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody toMycoplasma pneumoniae was produced.M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis ofM. pneumoniae infection in man. These studies were supported by Grants Al-15748 from the National Institute of Allergy and Infectious Diseases, and GM20138-07 from the National Institutes of Health, Bethesda, MD.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Monoclonal antibodies, L-35 and L-36, define novel T cell activation antigens

Two novel T cell specific activation antigens were characterized and were defined by monoclonal antibodies developed against mitogen-stimulated human T cells. These antigens, designated as L-35 and L-36 were expressed on both the CD 4(Leu-3) and the CD 8(Leu-2) subsets of activated but not resting T cells. Moreover these antigens were not expressed on a number of T, B, and myeloid tumor cell lines. L-35 and L-36 were expressed on interleukin 2 (IL 2)-dependent cloned T cell lines, and were weakly expressed on the T cell tumor line, HSB-2. L-35 was expressed on granulocytes and a small subset of thymocytes. SDS-PAGE analysis of 125I-labeled lysates from phytohemagglutinin (PHA)-activated T cells demonstrated that L-35 existed as a complex of 32,000 and 97,000 dalton polypeptides under reducing and nonreducing conditions. Anti-L-36 immunoprecipitated a 90,000 dalton structure from PHA-activated cell lysates prepared with CHAPS detergent. When immunoprecipitates were analyzed from [35S]methionine labeled lysates, anti-L-35 precipitated only the 97,000 dalton component, suggesting that the 32,000 dalton subunit of L-35 complex was not synthesized by the activated cell population. Furthermore anti-L-35 did not immunoprecipitate a 32,000 dalton component from 125I-labeled lysates of anti-Leu-4 or Con A-activated cells, suggesting that the 32,000 dalton component of the L-35 complex may represent a subunit of PHA. The 32,000 dalton protein could not, however, be precipitated from cells incubated with PHA for less than 1 day. These results suggested that anti-L-35 recognizes a 97,000 dalton structure expressed on activated T cells, and that upon activation by PHA, becomes associated with a subunit of this mitogen.     

Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization

The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation.     

Monoclonal antibodies specific for beta-adrenergic ligands

After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, eight clones were obtained secreting anti-alprenolol antibodies as characterized by means of an ELISA. Four of these were subcloned and were studied further. The association constant for alprenolol ranged from 1.9 X 10(6) M-1 to 24 to 10(6) M-1. Competitive inhibition of [3H]-l-dihydroalprenolol binding revealed cross-reactivity with beta-adrenergic ligands, with a higher avidity for antagonists than for agonists. Two of the antibodies had a higher affinity for the l-isomer than for the d-isomer. The most stereospecific of these antibodies showed only affinity for beta-adrenergic antagonists and for the agonist isoproterenol. The other recognized both beta-adrenergic antagonists and agonists; it also showed an increase in tryptophan fluorescence after ligand binding. This property was used for the physicochemical study of the hapten-antibody interaction.     

Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types

A 90-kD lymphocyte surface glycoprotein, defined by monoclonal antibodies of the Hermes series, is involved in lymphocyte recognition of high endothelial venules (HEV). Lymphocyte gp90Hermes binds in a saturable, reversible fashion to the mucosal vascular addressin (MAd), a tissue-specific endothelial cell adhesion molecule for lymphocytes. We and others have recently shown that the Hermes antigen is identical to or includes CD44 (In[Lu]-related p80), human Pgp-1, and extracellular matrix receptor III-molecules reportedly expressed on diverse cell types. Here, we examine the relationship between lymphoid and nonlymphoid Hermes antigens using serologic, biochemical, and, most importantly, functional assays. Consistent with studies using mAbs to CD44 or Pgp-1, mAbs against five different epitopes on lymphocyte gp90Hermes reacted with a wide variety of nonhematolymphoid cells in diverse normal human tissues, including many types of epithelium, mesenchymal elements such as fibroblasts and smooth muscle, and a subset of glia in the central nervous system. To ask whether these non-lymphoid molecules might also be functionally homologous to lymphocyte homing receptors, we assessed their ability to interact with purified MAd using fluorescence energy transfer techniques. The Hermes antigen isolated from both glial cells and fibroblasts--which express a predominant 90-kD form similar in relative molecular mass, isoelectric point, and protease sensitivity to lymphocyte gp90Hermes--was able to bind purified MAd. In contrast, a 140-160-kD form of the Hermes antigen isolated from squamous epithelial cells lacked this capability. Like lymphocyte binding to mucosal HEV, the interaction between glial gp90Hermes and MAd is inhibited by mAb Hermes-3, but not Hermes-1, suggesting that similar molecular domains are involved in the two binding events. The observation that the Hermes/CD44 molecules derived from several nonlymphoid cell types display binding domains homologous to those of lymphocyte homing receptors suggests that these glycoproteins represent a novel type of cell adhesion/recognition molecule (H-CAM) potentially mediating cell-cell or cell-matrix interactions in multiple tissues.     

Identification of murine germinal center B cell subsets defined by the expression of surface isotypes and differentiation antigens

Germinal centers (GCs) are inducible lymphoid microenvironments that support the generation of memory B cells, affinity maturation, and isotype switching. Previously, phenotypic transitions following in vivo B cell activation have been exploited to discriminate GC from non-GC B cells in the mouse and to delineate as many as seven distinct human peripheral B cell subsets. To better understand the differentiative processes occurring within murine GCs, we sought to identify subpopulations of GC B cells corresponding to discrete stages of GC B cell ontogeny. We performed multiparameter flow-cytometric analyses of GC B cells at consecutive time points following immunization of BALB/c mice with SRBC. We resolved the murine GC compartment into subsets based on the differential expression of activation markers, surface Ig isotypes, and differentiation Ags. Class-switched and nonswitched GC B cells emerged contemporaneously, and their relative frequencies remained nearly constant throughout the GC reaction, perhaps reflecting the establishment of a steady state. A significant percentage of the nonswitched B cells with a GC phenotype exhibited surface markers associated with naive B cells, including CD23, surface IgD, and high levels of CD38 consistent with either prolonged recruitment into the GC reaction or protracted expression of these markers during differentiation within the GC. Expression of the activation marker BLA-1 was dynamic over time, with all GC B cells being positive early after immunization, followed by progressive loss as the GC reaction matured into the second and third week. Implications of these results concerning GC evolution are discussed.     

The murine IL 2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells

The properties of three distinct rat monoclonal antibodies, designated 3C7, 7D4, and 2E4, to the murine IL 2 receptor have been compared in binding, biochemical, and functional assays. 3C7 appears to define an epitope near or identical to the IL 2-binding site of the receptor, because 3C7 inhibited the binding of radiolabeled IL 2 to CTL-L cells and because unlabeled IL 2 inhibited the binding of FITC-3C7 to CTL-L cells. 7D4 and 2E4 had no effect on IL 2 binding. Competitive antibody-binding studies confirmed that the epitope seen by 3C7 was distinct from the epitope(s) seen by 7D4 and 2E4. Sequential immunoprecipitation studies demonstrated that all three antibodies were reactive with the same molecular species, and that each precipitated identical components of 20,000 to 25,000 daltons, 50,000 to 60,000 daltons, and 100,000 to 120,000 daltons from the surface of CTL-L. FACS studies demonstrated a quantitatively and qualitatively identical cell distribution for the antigen defined by each antibody. They failed to stain more than 95% of resting lymphocytes, but were strongly reactive with Con A T blasts and substantially less reactive with LPS B blasts. Unlabeled IL 2 was also able to inhibit the binding of FITC-3C7 to LPS B cell blasts, suggesting the presence of IL 2-binding sites on activated B cells. Each antibody inhibited IL 2-driven proliferation of HT2 or CTL-L cells. 3C7 and 7D4 were more potent inhibitors of proliferation than was 2E4, and the combined use of 3C7 and 7D4 resulted in greater levels of inhibition of proliferation than that shown from the use of either antibody alone. Collectively, the results support the hypothesis that these antibodies detect two distinct functional regions of the IL 2 receptor.     

Monoclonal antibodies specific for v-abl- and c-abl-encoded molecules.

Monoclonal antibodies specific for regions of the transforming protein of Abelson murine leukemia virus were prepared. Antibodies directed against the kinase domain inhibited the autophosphorylation of v-abl proteins, and all of the antibodies reacted with the products of the murine and human c-abl loci.     

Tracking murine gammaherpesvirus 68 infection of germinal center B cells in vivo

Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency - which should facilitate studies to track MHV68 latently infected cells at late times post-infection.     

Monoclonal antibodies against rat Leydig cell surface antigens

Monoclonal antibodies (MAbs) directed against the Leydig cell surface may be used to identify this cell in testicular preparations. Collagenase-dispersed adult rat interstitial cells were fractionated on Percoll density gradients, and Leydig cell-enriched fractions were used to prepare MAbs. Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF) on isolated testicular cells and immunocytochemical localization on paraffin sections of adult testes. In density gradient fractions, immunoglobulin (Ig) M MAbs (LC-1C6 and LC-6H6) labeled the surface of cells possessing the morphological characteristics of Leydig cells. The density gradient profiles of MAb-binding activity observed by IIF and ELISA were parallel with the Leydig cell distribution as determined by [125I]-human chorionic gonadotropin (hCG) binding, testosterone response to hCG in vitro, 3 beta-hydroxysteroid dehydrogenase histochemistry and electron microscopy. The MAbs prominently labeled most interstitial cells in sections, but there was little or no labeling of connective tissue, endothelial or seminiferous tubule cells. Both MAbs recognized components of Mr 58,000 in Western blots of Leydig cell-enriched extracts. The results indicate that LC-1C6 and LC-6H6 recognize antigens on the Leydig cell surface that are not present on other isolated testicular cells from the adult rat. These MAbs are specific markers of the Leydig cell in situ and in vitro.     

Immunobiology of chicken germinal center: II. Accumulation of apoptotic cells within the germinal center

The germinal center (GC) develops after antigen stimulation and is thought to occur at the site of various immune responses. We observed apoptotic cells within the GC using in situ end labeling (TUNEL), small amount DNA ladder assay, and RT-PCR analysis of Bcl-2 mRNA expression. Apoptosis was detected within GCs at all phases of the GC reaction by both TUNEL and DNA ladder assays. The number of TUNEL⁺ nuclei within the GC did not increase over the course of the GC reaction. However, the density of DNA in the ladder assay was higher in later-phase GCs. Bcl-2 mRNA expression was detected within GCs during the early phases of the GC reaction. These results indicate that accumulation of apoptotic cells and rescue from apoptosis occur within chicken GCs. In the present paper, the reasons for the accumulation of apoptotic cells will be discussed.This work was supported by Grants-in-Aid for Scientific Research (Nos. 11670322 and 10306017) from the Ministry of Education, Science, Sport and Culture, and the Ministry of Agriculture, Forestry and Fisheries of Japan (Special Scientific Research and Pioneering Research Project in Biotechnology), as well as from the Bio-oriented Technology Research Advancement Institution (BRAIN)     

Aborted germinal center reactions and B cell memory by follicular T cells specific for a B cell receptor V region peptide

A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.     

Monoclonal antibodies specific for keratan sulfate detect epithelial-associated carbohydrates.

Monoclonal antibodies that specifically recognize epitopes on keratan sulfate glycosaminoglycans were used in this study to identify carbohydrate epitopes associated with many, but not all, types of epithelial cells. Immunoreactive cells included: keratinocytes, sebaceous gland cells, eccrine sweat gland duct cells, salivary gland excretory duct cells, colon adenocarcinoma cells, embryonic chick lung epithelial cells, embryonic chick mesonephric and metanephric kidney epithelial cells, and selected embryonic chick neural tube cells. Depending upon the type of epithelium, epitopes were located either within the cytoplasm or were located on cell surfaces. These epitopes were shared by cells from both human and chick tissues, indicating the absence of species specificity. Not all anti-keratan sulfate antibodies were equally effective in identifying epithelial-associated epitopes. One of the seven antibodies employed in this study failed to detect epitopes in almost all epithelial tissues studied. Of the remaining six antibodies, three were more effective than the others in recognizing epithelial-associated epitopes. These data indicate that carbohydrates that are typically associated with extracellular matrix can also be associated with epithelial cells, but in a form that is not necessarily related to extracellular matrix. These antibodies should prove to be useful in studies of the development of epithelial cells and tissues.     

Monoclonal antibodies specific for African swine fever virus proteins.

We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles.     

Monoclonal antibodies recognizing cell surface antigens in Drosophila melanogaster

Monoclonal antibodies have been made against cell surface antigens from Drosophila melanogaster, as probes for “differentiation antigens.” The immunogens used were 0–16 hr embryonic cells and mass isolated imaginal discs. The tissue distributions of the antigens recognized by three antiembryonic antibodies and two antiimaginal disc antibodies have been defined by indirect immunofluorescent (IIF) assays on differentiated embryonic cell cultures and on dissected third instar larval organs. These antigens fall into two major categories being either ubiquitous or tissue-limited in distribution. In indirect radioimmunoassays against 12 Drosophila cell lines the antigens showing ubiquitous tissue distributions were detectable on all cell lines whereas the tissue-limited antigens were absent from some cell lines. Such a screen against cell lines therefore provides a straightforward means of identifying antibodies against nonubiquitous antigens. One antibody recognizing a tissue-limited antigen was detected as muscle-specific by IIF assays on differentiated embryonic cell cultures. The second tissue-limited antigen was found to label basement membranes, by IIF assays against third instar larval organs. The temporal distribution of the antigens during embryogenesis (3–21 hr) has been studied by indirect radioimmunoassay. In this respect the antigens fall into three classes: (1) abundant throughout embryogenesis (a ubiquitous antigen), (2) present throughout embryogenesis but increasing markedly in abundance between 12 and 15 hr (two ubiquitous antigens and the muscle-specific antigen), and (3) absent early in development but appearing at about 12 hr postfertilization (the tissue-limited, basement membrane antigen).