MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive monoclonal antibodies.

mAb to human and mouse MHC molecules were tested for binding to blood or spleen cells of various nonmammalian vertebrates by immunofluorescence and flow cytometry. Those that bound were used to immunoprecipitate cross-reactive molecules from biosynthetically or cell surface-labeled spleen or blood cells. In addition, mAb to human MHC molecules were screened by Western blots. As expected from the results with xenoantisera, there were few mAb that cross-reacted, and many of these cross-reactions were not specific for MHC-like molecules. Less than 10% of the mAb tested bound to the cells of any particular species, with very few positive for more than one species. Of those mAb that bound cells, many failed to precipitate any radioactive bands, and most bands precipitated were not recognizable as MHC-like molecules. Five mAb reacted with Xenopus class II, one of which also immunoprecipitated axolotl class II. Another of these reacted with a candidate for class II in the lamprey, but this molecule had features unlike those expected for mammalian class II molecules. Four other mAb reacted with candidate molecules. in the trout and shark. None of the mouse alloantibodies immunoprecipitated nonmammalian vertebrate MHC-like molecules. In contrast to the results with most xenoantisera, the mAb cross-reacting with amphibian class II molecules recognized a number of different linear epitopes on the surface of the polymorphic non-Ig beta 1 domain of class II molecules. Few mAb recognized bands in Western blots of nonmammalian vertebrate cells and the candidate molecules from fish had features different from known mammalian MHC molecules.     

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.     

Human granulocyte surface molecules identified by murine monoclonal antibodies

We have investigated the nature of the antigens recognized by four classes of mouse anti-human monoclonal antibodies that characteristically reacted with neutrophilic granulocytes and their precursor cells, but not with monocytes or other normal hemopoietic cells. The antigenic targets of the majority (9/12) of the independently isolated monoclonal antibodies were present on two surface glycoproteins (Mr 145,000 and 105,000) and glycolipids. This antigen(s) was also detected on granulocyte precursor cells, including the bone marrow granulocyte/monocyte progenitor cells (CFU-GM). The same antigen(s) detected by these monoclonal antibodies was also present in non-hemopoietic cell lines (colon carcinoma and neuroblastoma). Three other antigens, defined by monoclonal antibodies AHN-8, L12.2, and L13.1 and present on granulocytes and their mid-late precursor cells, could not be identified as proteins but were detected in a protein-free glycolipid extract of these cells. The diversity of the antigens was confirmed by cross-competition experiments and by the identification of their different patterns of reactivity with cell lines and bone marrow cells.     

Binding of hyaluronic acid to lymphoid cell lines is inhibited by monoclonal antibodies against Pgp-1

Recent biochemical and sequence data suggest a possible relationship between Pgp-1 (identical to CD44/Hermes 1/p85) and a hyaluronic acid-binding function. Here, we have studied the hyaluronic acid-binding activity of a series of murine hematopoietic cell lines using several assays: cell aggregation by hyaluronic acid, binding of fluorescein-conjugated hyaluronic acid, and cell adhesion to hyaluronic acid-coated dishes. Certain Pgp-1-positive T and B cell lines show hyaluronic acid binding that is highly specific and is not competed for by other glycosaminoglycans. Monoclonal antibodies against Pgp-1, but not antibodies against other major cell surface glycoproteins, inhibited hyaluronic acid-induced cell aggregation and cell adhesion to hyaluronic acid-coated dishes. Additionally, some anti-Pgp-1 antibodies inhibited binding of fluorescein-hyaluronic acid to hyaluronic acid-binding lines. We found no Pgp-1-negative lines that bound, but many Pgp-1-positive cell lines did not bind hyaluronic acid. Two Pgp-1-positive thymomas that did not bind hyaluronic acid were induced by phorbol ester to bind hyaluronic acid with the same specificity as other hyaluronic acid-binding lines. Normal hematopoietic cells, including those which express high levels of Pgp-1, such as bone marrow myeloid cells and splenic lymphocytes, showed no detectable hyaluronic acid-binding activity. We discuss several models that might account for these observations: (1) the hyaluronic acid receptor is Pgp-1, but it normally exists in an inactive state; (2) hyaluronic acid receptors are a subset of a family of molecules recognized by anti-Pgp-1 antibodies; (3) the hyaluronic acid receptor is not Pgp-1, but is closely associated with Pgp-1 on the surface of cells which express hyaluronic acid-binding activity.     

OKT4+ cytotoxic T cells can lyse targets via class I molecules and can be blocked by monoclonal antibody against T4 molecules

Human cytotoxic T lymphocytes (CTL) have been shown to recognize either class I or class II major histocompatibility (MHC) products. This recognition has been correlated with the expression of OKT antigens on the surface of the CTL. Thus, OKT4+ CTL have been shown to be reactive with class II products, whereas OKT8+ effectors recognize class I molecules. In this study, responder cells were separated according to their OKT4 or OKT8 cell surface phenotype on a fluorescence-activated cell sorter (FACS). The OKT4+ subsets were stimulated with an LCL mutant that did not express DR and MB/MT but did express SB and class I antigens. After 7 days in culture, the activated subsets were tested on a panel of class I matched or mismatched targets. The cytotoxicity observed could be correlated with the presence of matched class I antigens. In addition, monoclonal antibody (MCA) W6/32, directed at a monomorphic determinant on HLA-A and -B molecules, blocked lysis. Furthermore, six OKT4+ CTL clones were derived from the OKT4+ bulk cultures; three clones were found to be directed at class I molecules whereas the other three recognized class II determinants. The ability of these clones to lyse their relevant targets was blocked by OKT4 MCA, raising questions as to the role of the T4 molecule in antigen class-specific CTL recognition.     

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.     

Blockade of Fc receptor-mediated binding to U-937 cells by murine monoclonal antibodies directed against a variety of surface antigens

The effect of murine IgG hybridoma antibodies directed against leukocyte antigens on the Fc receptor function of human cells was studied. For this purpose, the specific binding of 125I-labeled monomeric human IgG1 to a macrophage-like cell-line (U-937) was quantitated before and after incubation in the presence of murine monoclonal hybridoma antibodies. Four monoclonal hybridoma antibodies (A1G3, 23D6, 4F2, and 3A 10), each of which binds to different antigens on the surface of U-937 cells, rapidly and potently inhibited the specific binding of labeled IgG1 to these cells. Inasmuch as inhibition was mediated only by IgG antibodies with an intact Fc fragment and antibody activity against surface antigens found on U-937, inhibition appears to have resulted from the formation of a three-component complex composed of antibody bound by its Fab portion to antigen and by its Fc fragment to a Fc receptor. Equilibrium binding studies performed on treated cells confirmed that reduced Fc receptor-mediated binding was due to a reduction in the number of available receptors. Binding studies employing double isotope labeling methods demonstrated that about 0.5 to 1.0 Fc receptor was blocked for each molecule of intact antibody bound to a U-937 cell. Using several techniques, it was shown that most of the monoclonal antibody bound to cells and the Fc receptors blocked by antibody remained on the cell surface despite incubation at 37 degrees C for 3 hr. Thus, the loss of receptor function observed in these experiments was almost exclusively due to reversible receptor blockade rather than receptor internalization or degradation. The antibodies identified in these studies also markedly inhibited Fc receptors on one other human cell line (HL-60) as well as those on normal human peripheral blood monocytes.     

Generation of monoclonal antibodies directed against organ-specific endothelial cell surface determinants.

Organ-specific determinants expressed on the luminal surface of vascular endothelia are often unstable when cells are removed from their normal tissue environment and grown in culture. Unspecific endothelial cells of large vessel origin [e.g., bovine aorta (BAEC)] can be modulated to express and preserve such determinants when they are grown on the extracellular matrix of the desired organ. Lung matrix-modulated BAEC were used here to generate MAb against lung-specific vascular endothelia. Immunization was accomplished with outside-out membrane vesicles obtained by incubating BAEC monolayers grown on lung matrix with a low-strength paraformaldehyde solution. In four of the six fusions performed, this active immunization was preceded by passive immunization with mouse antiserum directed against membrane vesicles from BAEC grown on plastic. Among the growing hybrids, 7.6% secreted MAb that bound efficiently to both BAEC grown on lung-derived matrix and BAEC grown on plastic, while 3.5% (50) secreted MAb that bound primarily to BAEC grown on lung matrix. The fusion data show that only a passive/active immunization protocol yielded MAb directed against lung-specific endothelia. For example, MAb 6D3 and 5F5 selectively recognized endothelia from small- and medium-sized venules of bovine lungs, but failed to react with endothelial cells in other organs and tissues.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

Plasma membrane protrusions mediate host cell–cell fusion induced by Burkholderia thailandensis

Cell–cell fusion is important for biological processes including fertilization, development, immunity, and microbial pathogenesis. Bacteria in the pseudomallei group of the Burkholderia species, including B. thailandensis, spread between host cells by inducing cell–cell fusion. Previous work showed that B. thailandensis-induced cell–cell fusion requires intracellular bacterial motility and a bacterial protein secretion apparatus called the type VI secretion system-5 (T6SS-5), including the T6SS-5 protein VgrG5. However, the cellular-level mechanism of and T6SS-5 proteins important for bacteria-induced cell–cell fusion remained incompletely described. Using live-cell imaging, we found bacteria used actin-based motility to push on the host cell plasma membrane to form plasma membrane protrusions that extended into neighboring cells. Then, membrane fusion occurred within membrane protrusions either proximal to the bacterium at the tip or elsewhere within protrusions. Expression of VgrG5 by bacteria within membrane protrusions was required to promote cell–cell fusion. Furthermore, a second predicted T6SS-5 protein, TagD5, was also required for cell–cell fusion. In the absence of VgrG5 or TagD5, bacteria in plasma membrane protrusions were engulfed into neighboring cells. Our results suggest that the T6SS-5 effectors VgrG5 and TagD5 are secreted within membrane protrusions and act locally to promote membrane fusion.     

Analyses of epididymal sperm surface antigens by monoclonal antibodies against hamster spermatozoa

BALB/c mice were immunized with spermatozoa from cauda epididymides of hamsters and the immune spleen cells were fused with mouse myeloma cells (P3U1). Seven hybridomas (GHS-1,-2,-3,-4,-5,-6, and -7) that produced monoclonal antibodies (Mabs) binding to the epididymal spermatozoa were established. Three Mabs (GHS-3,-4, and -6) were IgM and the other four were IgG1. All Mabs reacted to hamster spermatozoa from cauda epididymides but none of the Mabs except GHS-5 and -7 reacted to spermatozoa in testis. GHS-5 and -7 Mabs bound to the acrosome region of spermatozoa in both testis and epididymis. The antigens corresponding to GHS-2, -4, and -6 Mabs appeared to be excreted from epithelial cells of caput epididymis, while those to GHS-1 and -3 Mabs seemed to be produced in cauda epididymis. Both groups of the antigens bound to the surface of spermatozoa during their epididymal transit. Immunoblotting analyses of epididymal fluid showed that the antigen epitopes corresponding to GHS-1,-2,-3,-4, and -6 Mabs were distributed to multiple components with different molecular weights ranging from over 100 to 25 kd. The distribution patterns of the epitopes corresponding to GHS-1 and -3 Mabs and GHS-2,-4, and -6 Mabs were very similar, respectively, but each group pattern was quite different from each other. GHS-5 Mab reacted to a component of sperm extract with a molecular weight of around 94 kd, while GHS-7 Mab failed to recognize any components transblotted.     

Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18

We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Select host cell proteins coelute with monoclonal antibodies in protein A chromatography

The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbs-HCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb.     

Schistosoma mansoni: detection by monoclonal antibody of a 22,000-dalton surface membrane antigen which may be blocked by host molecules on lung stage parasites

Monoclonal antibody M.2 binds to the surface membranes of cercariae and developing schistosomula. This antibody was generated from mice immunized with membrane-enriched extracts of mechanically transformed schistosomula. The antigen detected by M.2 was shown to persist on developing schistosomula for at least 96 hr post-transformation. M.2 also bound to the surface of living, cultured lung worms but not to freshly harvested lung worms. The ability of M.2 to bind to cultured lung worms coincided with the loss of host H-2 from the parasite surface. The apparent m.w. of the antigen was 22,000; an antigen with the same apparent m.w. was immunoprecipitated from cercariae, schistosomula, lung worms, and adult worms.     

Surface antigenic analysis of rosette-forming cells in rat thymus--using monoclonal antibodies against T cell surface markers.

We have assumed that rat thymocytes capable of rosette-formation with guinea pig erythrocytes were double positive (CD4+8+) cells. Therefore surface-binding erythrocytes on rosette-forming lymphocytes (RFLs), separated in highly pure state, were eliminated by a hypotonic shock. The surface markers of RFLs, without surface erythrocytes, were analyzed by flow cytometry using two kinds of monoclonal antibodies (anti-CD4, anti-CD8) labelled with different fluorescence materials. About 96.2% of cells in the RFL population were sorted in two areas, into which double positive cells were separated. This result substantiates the assumption mentioned above. It was, furthermore, discussed that the RFLs without surface erythrocytes would be one of the valuable materials for disclosing the mechanisms of differentiation and maturation from double positive thymocytes to single positive ones.     

Synchronized onset of nuclear and cell surface modifications in U937 cells during apoptosis

In this study we investigated the relationship between nuclear and cell surface modifications (i.e. blebbing, phosphatidylserine [PS] and sugar residues exposure) in a monocytic cell line, U937, during apoptosis induced by oxidative stress (1 mM H2O2) or inhibition of protein synthesis (10 microg/ml puromycin). Dying cells were simultaneously observed for nuclear modifications, presence of superficial blebs and plasma membrane alterations. Morphological analysis performed by conventional fluorescence microscopy, or by transmission and scanning electron microscopy showed that the courses of nuclear and membrane alterations occured concomitantly, but the phenotype was dependent on the stage of the apoptotic process and the type of apoptogenic inducer used. The progression of apoptosis in U937 cells beyond early stages resulted in the extensive formation of blebs which concomitantly lost some typical markers of apoptosis, such as PS and sugar residues. Therefore, the modality by which the nucleus condenses, or the amount and the pattern of distribution of PS on the cell surface were, for each cell line, strictly related to the apoptogenic inducer. The morphological data reported in the present paper should lead to a more precise quantification of apoptosis by improving the detection of apoptotic cells in vivo (i.e. in tissue, organs), which is a crucial point in the evaluation of efficiency of antiproliferative drugs, such as antiblastic or immunosuppressive compounds.     

Differential expression of cell surface antigens of canine keratinocytes defined by monoclonal antibodies

Nine monoclonal antibodies (MAb) directed against cell surface antigens of canine keratinocytes define distinct keratinocyte subpopulations owing to the differential expression of these antigens during the process of differentiation and depending on the tissue location of the cells. There was distinct antigenic heterogeneity between the different layers of stratified squamous epithelium and between stratified squamous epithelial of different tissue origin. Two MAb reacted only with antigens expressed by esophageal mucosa. Three MAb bound to antigens on keratinocytes of the suprabasilar and granular layers of stratified squamous epithelia, and they crossreacted with the transitional epithelial cells of the urinary tract. Two MAb reacted with antigens only expressed on differentiated cells, superficially located in the stratified squamous epithelium. The use of these MAb as markers for keratinocytes in studies on the characterization and differentiation of keratinocytes, as well as in tumor diagnosis and allograft transplantation, is discussed.