BLAST-2 [EBVCS], an early cell surface marker of human B cell activation, is superinduced by Epstein Barr virus

Activation of human B cells by pokeweed mitogen (PWM), protein A, anti-IgM, or EBV infection results in the expression of a new surface antigen, termed BLAST-2 [EBVCS]. This marker appears before the cells undergo blast transformation as assessed by the initiation of DNA synthesis and expression of the BLAST-1 antigen. Thus, the BLAST-2 [EBVCS] antigen is expressed on both activated and lymphoblastoid cells. The antigen is, in addition, restricted to B cells, as it is not found on cells of T or myeloid lineage derived from peripheral blood, cell lines, or neoplastic cells. However, it is readily detected on chronic lymphocytic leukemia cells of B cell origin and in the germinal centers of tonsils and lymph nodes. Like the BLAST-1 antigen, BLAST-2 [EBVCS] is expressed at a high level only on EBV-transformed B lymphoblasts and has a m.w. close to 45,000. Immunoprecipitation experiments show, however, that the two antigens are expressed on distinct populations of molecules.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.     

Human major histocompatibility complex class II invariant chain is expressed on the cell surface.

Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.     

Flow microfluorometric analysis of H-2L expression

The cell surface expression of H-2L, a major transplantation antigen, was compared by flow microfluorometry to the expression of products of H-2K and H-2D loci, using monoclonal antibodies. By this methodology, the ontogeny and tissue distribution of Ld antigens were found to be indistinguishable from those of the K and D antigens. In a reciprocal blocking assay, using fluorescein-labeled test reagents, it was shown that monoclonals anti-H-2.65 and anti-H-2.64 did not inhibit the binding of each other. These results suggest that the alloantigenic determinants H-2.64 and H-2.65 are located at distinct sites on Ld molecules. Quantitative comparisons using the fluorescein-labeled monoclonal reagents indicated that Ld molecules are expressed at 2- to 3-fold lower levels on the cell surface compared with K and D molecules. These findings give new credence to a "3-locus" model for the major histocompatibility complex of man and mouse, where H-2L and HLA-C share several homologies that are unique and distinguish them from the other histocompatibility loci.     

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.     

Phenotypic Analysis of New World Primate Mononuclear Cell Surface Antigens

We have applied a panel of monoclonal antibodies against antigens present on the surface of human mononuclear cells to the study of mononuclear cell surface antigens expressed by seven species of New World primates. Antibodies to the sheep erythrocyte receptor (OKT11a) to a thymocyte antigen (OKT10), to the I region of the major histocompatibility locus (OKIa), and to an antigen found on the surface of human monocytes (OKM1) cross-reacted with mononuclear cell surface antigens of most of the species studied. Antibodies to antigens which have been correlated with functional capabilities in the human system (OKT4, OKT5, OKT8, 3A1) were much less reactive with platyrrhine mononuclear cells. These reagents may be quite useful in studies of primate phylogeny and immunology.     

Heterogeneity of allogeneic restriction of human cytotoxic T cell clones specific for Epstein Barr virus

Clones of human cytotoxic T cells (Tc) specific for Epstein Barr virus (EBV) were isolated from peripheral blood lymphocyte (PBL) cultures stimulated repeatedly with autologous EBV-transformed lymphoblastoid cell line (LCL) cells in vitro. The method employed to clone EBV-specific Tc was a limiting dilution technique utilizing T cell growth factor (TCGF). The EBV specificity of Tc clones was determined by showing that they were significantly cytotoxic for autologous LCL cells but not for either autologous PBL or (natural killer-sensitive) K-562 cells. Eight EBV-specific Tc clones derived from a single donor exhibited distinct cytotoxic patterns against allogeneic LCL targets. Two clones were cytotoxic to LCL targets sharing both HLA-A26 and B15 antigens with effectors, and killing by two other clones was strongly restricted to autologous LCL cells. The four remaining clones showed cytotoxicities against various allogeneic LCL targets irrespective of HLA antigen expression. Eight EBV-specific Tc clones derived from a second donor also exhibited a wide spectrum of cytotoxicity to allogeneic LcL targets. We conclude that EBV-specific Tc, induced in vitro, consist of a number of clones with respect to restrictions imposed by the major histocompatibility complex. The determinants regulating these restrictions may include not only private HLA antigenic determinants that are defined by the HLA serotyping, but also undefined HLA antigenic determinants.     

Endocytosis of HLA and H-2 molecules on transformed murine cells measured by fluorescence dequenching of liposome-encapsulated carboxyfluorescein.

We have studied internalization of cell surface proteins encoded by genes of the human major histocompatibility complex (HLA) transferred into murine L cells, in comparison with mouse (H-2) histocompatibility determinants. This internalization was measured by the use of monoclonal antibodies directed at these determinants, to which were bound methotrexate- and carboxyfluorescein-containing liposomes covalently coupled to protein A. In addition to the effect of methotrexate on the cells, the technique of fluorescence self-quenching release was used to measure the kinetics of internalization for single cells using a flow cytofluorometer. The native and foreign gene products were internalized in an apparently identical manner. These techniques can be applied to cells expressing genes with altered or deleted segments thus providing a basis for the analysis of the effect of sequence modifications on the internalization of the encoded molecule.     

Cross-reactivity between major histocompatibility complex class II antigens in mice NOD and an islet antigen with 58 kDA molecular weight

The NOD mouse has been recently developed as a model for autoimmune insulin-dependent diabetes mellitus. The NOD mouse is further characterized by a genetic linkage with genes mapping within the major histocompatibility complex at the I-A locus. The present work demonstrates the presence in NOD mice of circulating autoantibodies specific for a 58 kDa antigen which is present in membrane extracts prepared from the murine insulin-secreting tumoral cell line Rin5F. This 58 kDa antigen shows a cross reactivity with NOD mouse class II antigens as indicated by its recognition by anti-I-A(NOD) monoclonal antibodies.     

Expression of a T cell receptor-like molecule on normal and malignant murine T cells detected by rat monoclonal antibodies to nonclonotypic determinants

A T cell receptor-like molecule with a dimer structure of 45 kilodaltons (Kd) under reducing and 90 Kd under nonreducing conditions was detected on the surface of two murine T lymphoma lines, EL-4 and MBL-2, by two rat monoclonal antibodies. The two antibodies seemed to react with different determinants on the same molecule. The antibodies did not react with the surface of normal T cells as tested by flow cytometric analysis of cell surface staining. Two-dimensional gel electrophoresis (IEF vs SDS-PAGE) and tryptic peptide analysis revealed the molecule to consist of two chains with different isoelectric points and different tryptic peptides. A conventional antiserum was raised against the heterodimer purified from EL-4 cells. The immune serum did not bind to the surface of normal T cells. However, the immune serum as well as the monoclonal antibodies immunoprecipitated the dimer molecules from detergent-solubilized normal thymocytes and spleen cells. The dimer molecule was detected on both immature and mature thymocytes. These results suggest that the antibodies detect non-clonotypic determinants on a T cell receptor-like protein. The determinants are masked on the surface of normal T cells, whereas they are exposed on the surface of at least two T lymphoma cell lines. Three polypeptides of 30 Kd, 25 Kd, and 15 Kd were also coprecipitated with the heterodimer from MBL-2 cells. These proteins may associate with the heterodimer and may be masking the antigenic determinants on normal T cells. The relationship between the heterodimer molecule described here and the T cell antigen receptor or the human T cell antigen 9.3 is still unknown.     

The interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. I. Analysis of the mechanism of binding

We studied the mechanism of binding of radiolabeled, monoclonal anti-H-2 antibodies to mouse spleen cells to determine the number of H-2 antigen molecules per cell. Equilibrium and kinetic data were analyzed in detail according to theoretical models developed for different modes of antibody binding. The results of binding experiments from three monoclonal IgG antibodies (36-7-5, anti-Kk; 27-11-13, anti-DbDd; and 11-4-1, anti-Kk) and their F(ab')2 and F(ab') fragments show that for the IgG and F(ab')2 from all three antibodies, the monovalently and bivalently bound states of the antibody co-exist in rapid equilibrium with one another on the cell surface, with the bivalent state predominating. We show that the relative proportions of the monovalently and bivalently bound species can be estimated from dissociation kinetics experiments, and that once the mode of antibody binding has been established, the density of H-2 determinants on the cell surface can be estimated from equilibrium-binding data. We conclude that the average numbers of H-2K and H-2D molecules on B10.A spleen cells are 5 X 10(4) and 1.1 X 10(5) molecules/cell, respectively.     

Class I major histocompatibility proteins are an essential component of the simian virus 40 receptor.

The class I molecules encoded by the major histocompatibility complex (MHC) present endogenously synthesized antigenic peptide fragments to cytotoxic T lymphocytes. We show here that these proteins are an essential component of the cell surface receptor for simian virus 40 (SV40). First, SV40 binding to cells can be blocked by two monoclonal antibodies against class I human lymphocyte antigen (HLA) proteins but not by monoclonal antibodies specific for other cell surface proteins. Second, SV40 does not bind to cells of two different human lymphoblastoid cell lines which do not express surface class I MHC proteins because of genetic defects in the beta 2-microglobulin gene in one line and in the HLA complex in the other. Transfection of these cell lines with cloned genes for beta 2-microglobulin and HLA-B8, respectively, restored expression of their surface class I MHC proteins and resulted in concomitant SV40 binding. Finally, SV40 binds to purified HLA proteins in vitro and selectively binds to class I MHC proteins in a cell surface extract.     

Differential expression of guinea pig class II major histocompatibility complex antigens on vascular endothelial cells in vitro and in experimental allergic encephalomyelitis

Previous studies have shown that vascular endothelial cells do not normally express major histocompatibility complex (MHC) Class II antigens either in vivo or in vitro. In this investigation it was found that endothelial in the central nervous system (CNS) of normal guinea pigs constitutively express MHC Class II antigens recognized by the monoclonal antibodies HLA-DR, 27E7, and MSgp8. This phenotype is retained when these CNS-derived endothelial cells are propagated in tissue culture. Furthermore, examination of CNS tissue taken from animals in the acute phase of chronic relapsing experimental allergic encephalomyelitis shows that additional epitopes of the MHC Class II antigen, detected by the monoclonal antibodies CI.13.1 and 22C4, are present during the diseased state. This study not only demonstrates constitutive expression of certain MHC Class II determinants by guinea pig endothelial cells, but also shows that other Class II determinants can be differentially expressed in certain disease states.     

T-cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system

Clearance of the neurotropic JHM strain of mouse hepatitis virus from the central nervous system was examined by the transfer of spleen cells from immunized donors. A T cell with the surface phenotype of Thy1.2+ CD4+ CD8- asialo-GM1+ Mac-1- was found to be necessary for viral clearance. The surface phenotype and adherence to nylon wool suggest that these cells are activated helper-inducer T cells. Adoptive transfer to congenic histocompatibility strains demonstrated the necessity for compatibility at the D locus of the major histocompatibility complex. The expression of the CD4 surface marker and the requirement for major histocompatibility complex class I were further studied by the transfer of cells to recipients treated with anti-CD4 or anti-CD8 monoclonal antibodies. Treatment of recipients with either the anti-CD8 or the anti-CD4 antibodies inhibited virus clearance from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system by CD8+ cells that recognize viral antigen in the context of the H-2Db gene product.     

HLA class l specific t lymphocyte clones with dual alloreactive functions

Four human T lymphocyte clones exhibiting proliferative responses to class I HLA antigens were isolated from an in vitro mixed lymphocyte culture (MLC). Three clones expressed the Leu-2⁺3^– phenotype and demonstrated proliferation in response to HLA-B8, while the fourth clone expressed the Leu-2^–3⁺ phenotype and proliferated in response to HLA-A2. These clones were also cytotoxic towards cells bearing the same target antigens. Blocking studies utilizing monoclonal antibodies demonstrated that proliferation was triggered by determinants on the class I molecule itself, and these determinants appear to be spatially close to those which determine serologic allospecificity. These findings support the concept that the class I molecules themselves are the weak MLC stimulating determinants previously mapped to the HLA-A and B regions of the major histocompatibility complex.Abbreviations used in this paper B-LCL B-lymphoblastoid cell line - cpm counts per minute - FCS fetal calf serum - HS human serum - ³H-TdR tritiated thymidine - IL-2 interleukin-2 - IL2-CM interleukin-2 containing conditioned medium - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MoAb monoclonal antibody - PBL peripheral blood mononuclear leukocytes - PLT primed lymphocyte test     

Demonstration of two allelic forms of the bovine T cell antigen Bo5 (CD5) and studies of their inheritance

Two monoclonal antibodies (mAbs), CC17 and IL-A67, which are specific for the bovine equivalent of the CD5 antigen, Bo5, were each found to react with the cells of some animals but not others. The cattle tested were all positive for one or both of the mAbs, but the level of expression on cells expressing both determinants was slightly lower than that on cells expressing either of the determinants on their own. Both mAbs precipitated an antigen of 67 kD. However, sequential immunoprecipitation experiments with cells that reacted with both mAbs demonstrated that the determinants are present on two different sets of molecules. These findings suggested that the mAbs recognize two co-dominantly expressed allelic forms of Bo5. This was confirmed in family studies, with groups of full- and half-sibling offspring of sires and dams of defined phenotypes. These experiments also showed that the gene encoding the Bo5 antigen is not linked to the major histocompatibility complex (MHC). The frequencies of the two alleles, which have been designated Bo5.1 and Bo5.2, in the cattle populations tested were 100% and 0%, respectively, in Bos taurus, and 10% and 90%, respectively, in Bos indicus.     

Monoclonal antibodies specific for human chromosome 5 obtained with a monochromosomal hybrid can be used to sort out cells containing the chromosome with a FACS

Using a human-mouse monochromosomal hybrid, BG15-6, that contains an intact human chromosome 5, we isolated four monoclonal antibodies, 2A10, 3H9, 5G9, and 6G12, as chromosome marker antibodies recognizing cell surface antigens specific for human chromosome 5. The binding patterns of these antibodies to BG15 subclones containing fragments of human chromosome 5 indicated that 2A10, 3H9, and 6G12 recognized the antigens produced by genes located on 5pterq22, and that 5G9 recognized the antigen produced by a gene located on 5q23. Cells containing human chromosome 5 were very effectively sorted in a fluorescence-activated cell sorter (FACS) using monoclonal antibody 6G12. This method for sorting cells containing human chromosome 5 or an appropriate fragment of this chromosome from among human-rodent hybrid cells should be very useful in studies on gene expression, gene cloning and gene mapping.by M. Trendelenburg     

Regulation of immune responses by I-J gene products. V. Heterogeneity of I-J gene products as detected by anti-I-J monoclonal antibodies

The products of I-region genes of the murine major histocompatibility complex (H-2) are intimately involved in the regulation of immune responses. In a number of antigen systems, suppressor T (Ts) cells and their factors (TsF) bear determinants of gene(s) that map to the I-J subregion of the H-2 complex. Suppression in one such system, poly(Glu50Tyr50)(GT), involves the interaction of multiple Ts subsets. Three monoclonal I-Jk-bearing GT-TsF have been identified that functionally differ from one another. We report the binding of these monoclonal GT-TsF to different anti-I-Jk monoclonal antibody columns. We find that these anti-I-Jk monoclonal antibodies display differing degrees of efficiency of GT-TsF binding. These data suggest a greater degree of heterogeneity of I-Jk gene products than has been proposed before.     

Mechanism of escape of endogenous murine leukemia virus emv-14 from recognition by anti-AKR/Gross virus cytolytic T lymphocytes.

It was previously shown that spleen cells from endogenous ecotropic murine leukemia virus emv-14+ AKXL-5 mice fail to stimulate an anti-AKR/Gross virus cytolytic T-lymphocyte (CTL) response in a mixed lymphocyte culture with primed C57BL/6 responder spleen cells, whereas spleen cells from AKXL strains carrying the very similar emv-11 provirus do stimulate a response (Green and Graziano, Immunogenetics 23:106-110, 1986). We wished to determine whether the lack of response with AKXL-5 spleen cells was at the level of recognition between effector cell and target cell and whether the relevant mutation was within the emv-14 provirus. It is shown here that EMV-negative SC-1 fibroblast cells transfected with the major histocompatibility complex class I Kb gene and infected with virus isolated from the AKXL-5 strain (SC.Kb/5 cells) were not lysed by H-2b-restricted anti-AKR/Gross virus CTL. SC.Kb cells infected with virus isolated from emv-11+ strains, however, were efficiently lysed by anti-AKR/Gross virus CTL, indicating that there is nothing intrinsic to EMV-infected SC.Kb cells that would prevent them from being recognized and lysed efficiently by anti-AKR/Gross virus CTL. Analysis of virus expression for the infected SC.Kb cells by XC plaque assay and by flow cytometry indicated that emv-14 virus expression for SC.Kb/5 cells was not significantly different from that for emv-11-containing SC.Kb/9 or SC.Kb/21 cells. These data show that the mutation responsible for the lack of CTL recognition and lysis is at the level of recognition between target cell and effector cell. Furthermore, these data strongly suggest that the mutation is within the emv-14 genome. Flow cytometry experiments with monoclonal antibodies against a number of viral determinants indicated that there was no gross mutation detectable in the viral determinants analyzed. The data suggest that the relevant mutation may be a point mutation or a small insertion or deletion within a coding sequence that is critical for CTL recognition.