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F W Symington

Publications and source records attributed to F W Symington.

29 records · Page 2Linked to original sources

Differential Ia antigen expression by autologous human erythroid and B lymphoblastoid cell lines.

Human erythroid precursors express Ia antigens that have serology, function, molecular nature, and genetic regulation that are largely unknown. To approach these issues, Ia+ and Ia- subclones of the HEL human erythroleukemia cell line (HEL-DR+ and HEL-DR-, respectively) and an autologous B lymphoblastoid line (B line) were isolated. These erythroid and lymphoid lines were compared with respect to their binding of monoclonal HLA-D subregion-specific antibodies, the ability to trigger in vitro alloproliferation, expression of class II molecules, and transcription of class II-related genes. Unlike the DP+/DQ+/DR+ B lines, HEL-DR+ differentially expressed DP and DR, but not DQ specificities. Also unlike the autologous B line, HEL-DR+ appeared to be unable to trigger primary or secondary allogeneic T cell proliferation, despite the presence of responder monocytes in these cultures and irrespective of lymphokine addition. HEL-DR+ expression of bona fide class II molecules similar to B line DR heterodimers was verified by two-dimensional gel electrophoresis of material immunoprecipitated from 125I-labeled cells. Northern blot analysis of cytoplasmic RNA from these lines indicated that differential class II gene transcription could readily explain the distinct, lineage-related Ia phenotypes of HEL-DR+ and B line. In addition, the lack of invariant chain and class II transcripts in HEL-DR- implied that expression of these unlinked genes in HEL cells is co-regulated.

Antigen-Antibody Reactions↗

Monoclonal antibody specific for lactosylceramide.

The mouse hybridoma line T5A7 was derived during studies aimed at mapping human myeloid differentiation antigens. The IgM antibody secreted by this line recognizes an antigen richly expressed on mature myelomonocytic cells and on a subpopulation of lectin-activated human T-lymphocytes (Andrews, R. G., Torok-Storb, B., and Bernstein, I. D. (1983) Blood 62, 124-132). In the present study, we have determined the specificity of T5A7 antibody to be directed to lactosylceramide (Gal beta 1----4Glc beta 1----1 Cer) based on direct and indirect binding assays using a variety of glycolipids with known structures. The antibody did not cross-react with glycolipids having an N-acetyllactosamine terminus, including lactoneotetraosylceramide lactonorhexaosylceramide (i antigen), and lactoisooctaosylceramide (I antigen). The possible contribution of ceramide to the reactivity of lactosylceramide with this antibody was also studied using lactosylceramide preparations having different fatty acid composition.

Animals↗

Hematopoietic subpopulations express cross-reactive, lineage-specific molecules detected by monoclonal antibody.

The molecular specificity of a rat anti-mouse monoclonal antibody for cell surface antigens expressed by T- and B-lymphocyte subsets, erythrocytes and polymorphonuclear neutrophils (PMN) was determined. The antibody reacts with B-lymphocyte-associated molecules which migrated as a sharp 48,000 mol. wt band on SDS-PAGE. The antibody reacts with heterogeneous thymocyte and PMN molecules with a predominant mol. wt of 52,000. The same antibody reacts with heat-stable, amphipathic, organic-solvent-soluble erythrocyte molecules of mol. wt 35,000-40,000 present in Folch upper-phase ganglioside fractions, and evidence is presented that the determinant is protein-defined. Thus, a single monoclonal reagent which recognizes distinct, lineage-specific cell surface proteins on erythroid, lymphoid and myeloid elements may be used to probe not only the characteristic patterns of development of these hematopoietic subsets, but also the biochemical functions of the protein antigens themselves. In the case of B- and T-lymphocytes, such functions may extend to involvement in ligand-induced maturation and repertoire selection.

Animals↗

Fine specificity of a monoclonal anti-testicular cell antibody for glycolipids with terminal N-acetyl-D-glucosamine structure.

The specificity of a mouse anti-testicular cell monoclonal antibody, J1, was investigated. Previous studies suggested that N-acetyl-D-glucosamine (GlcNAc) was a constituent of the determinant recognized by J1. When the antibody was tested against a variety of purified glycolipids containing this saccharide in terminal, penultimate or internal positions, J1 reacted only with species expressing terminal GlcNAc. The influence of oligosaccharide chain length, branch substitution, and haptenic valence on J1 binding was examined using glycolipids prepared by a weak acid hydrolysis and exoglycosidase digestion of bovine I-active ganglioside. Degree of binding was inversely proportional to chain length and was proportional to hapten valence. Failure of J1 to bind partially deglycosylated transferrin implied binding preference for GlcNAc beta 1----3Gal over GlcNAc beta 1----2Man. Immunofluorescence analysis of J1 binding to human neutrophils failed to detect lactotriosylceramide on their surface, although this glycolipid has previously been isolated from these cells, suggesting that this structure exists in a cryptic or intracellular form. Binding results were consistent with J1 having low affinity for GlcNAc or GlcNAc beta 1----3Gal on a variety of lacto-series glycolipids.

Acetylglucosamine↗

Biochemical characterization of Ia antigens encoded by the RT1.B and RT1.D loci in the rat MHC.

Ia antigens in rats are genetically associated with typical MHC-linked immune response (Ir) genes. One rat Ir gene (IR-GLT) has recently been mapped to the RT1.D locus in a rat MHC recombinant, WRC. We have studied the expression of Ia antigenic determinants in WRC and its parental strains BN and WRA using a panel of monoclonal antibodies. Our results suggest that the inheritance of IR-GLT corresponds with the inheritance of I-E-like, but not I-A-like, antigens in the WRC rat. These observations were confirmed when WRC I-E-like antigens were analyzed by two-dimensional gel electrophoresis. We propose that RT1.D shares both functional and antigenic homologies with the mouse I-E subregion.

Animals↗

Lyb-8.2: A new B cell antigen defined and characterized with a monoclonal antibody.

A DBA/1 B10.D2-specific monoclonal antibody (CY34) is described which defines a new murine B lymphocyte differentiation antigen designated Lyb-8.2. The ontogeny, strain distribution, and cell-surface density of the antigen were studied by radioimmunoassay and by fluorescence-activated cell sorter (FACS) analysis. Lyb-8.2 appears to be expressed on pre-B cells and on all mature B lymphocytes. Lyb-8.2 molecules immunoprecipitated from surface labeled B10.D2 spleen cells migrated in polyacrylamide gels with an apparent mol. wt. of 95,000-105,000 daltons and were bound by lentil lectin. The expression of Lyb-8.2 is controlled by a locus on chromosome 7 that is closely linked to Gpi-1 and RP-2. Added Lyb-8.2-specific antibody did not measurably impair B lymphocyte function in several in vitro systems studied.

Animals↗

Inhibition of T cell activation in vivo with mixtures of monoclonal antibodies specific for I-A and I-A/E molecules.

(CBA x B6)F1 (Iak x Iab) T cells were activated to sheep erythrocytes in irradiated F1 mice in the presence of various monoclonal anti-Ia reagents and then tested for their capacity to collaborate with B cells from B10.BR (I-Ak, I-Ek) (kk), B10.A(4R) (kb), and B10 (bb) mice. Anti-I-Ak antibodies blocked the generation of help for B10.A(4R) B cells, but not B10.BR or B10 B cells. An anti-I-Ab antibody blocked help for B10 B cells, but not for B10.BR or B10.A(4R) B cells. An antibody (Y-17) specific for I-Ak/Ek and I-Ab/Ek molecules, but not for I-Ak or I-Ab molecules, failed to impair the generation of help for B10.BR, B10.A (4R), or B10 B cells. In marked contrast to injecting each antibody separately, a mixture of anti-I-Ak and anti-I-Ak,b/Ek (Y-17) antibodies virtually abolished the generation of help for B10.BR B cells. A mixture of anti-I-Ak and anti-I-Ab antibodies effectively blocked help for (4R x B10)F1 B cells, i.e., cells expressing hybrid I-A molecules. These two antibodies only marginally impaired help for (CBA x B6)F1 B cells. To block help for (CBA x B6)F1 B cells required selection in the presence of a cocktail of anti-I-Ak, anti-I-Ab, and anti-I-Ak,b/Ek antibodies. The implications of these findings are discussed.

Animals↗

A monoclonal antibody discriminating between subsets of T and B cells.

A description is given of a rat anti-mouse hybridoma antibody, JIId, which reacts with erythrocytes, neutrophils, greater than 90% of thymus cells, and most B cells. JIId does not have detectable activity for mature T cells, pluripotential stem cells, platelets, or cells of the monocyte-macrophage lineage. Although the JIId antigen is present on 90 to 95% of typical small B lymphocytes, pretreatment of spleen cells with JIId plus complement has no effect on secondary IgG antibody responses; by contrast, primary IgM responses and proliferative responses to lipopolysaccharide are substantially reduced. Unlike the precursors of IgG antibody-producing cells (AFC), IgG AFC per se are strongly JIId-positive. Rapid acquisition of the JIId antigen also applied to the early progeny of pluripotential stem cells.

Animals↗

A two-signal mechanism for the induction of cytotoxic T lymphocytes.

Depletion of adherent cells from stimulator and responder lymphocytes by a single filtration through nylon wool columns led to complete abrogation of the cytotoxic response to the stimulating alloantigen. Cytotoxic responses were restored by adding anti-Thy-1 + complement-greated normal peritoneal exudate cells (PEC) syngeneic or allogeneic to the responding population. Alternatively, the response could be reconstituted with costimulator, a lymphokine obtained by stimulation of spleen cells with concanavalin A. Costimulator was not itself cytotoxic and induced few or no cytotoxic T lymphocytes (CL) in the absence of stimulator cells. Costimulator was also more efficient than allogeneic PEC, which in turn were more efficient than syngeneic PEC, in reconstituting the cytotoxic response. The number of CL produced to the activating alloantigen was shown to increase with increasing concentration of costimulator. More interestingly, in the presence of a relatively high concentration of costimulator, CL were also activated to target cells that differ in H-2 haplotype from the stimulating alloantigen. Lysis of the third-party target cells could not be inhibited by cold targets syngeneic to the activating alloantigen. A clonal assay for cytotoxic precursors was used to confirm that CL for the activating alloantigen and CL for the third-party H-2 antigens were derived from different progenitors. Only about 37% of the cytotoxic clones produced were specific for the activating alloantigen. These observations are explained in terms of a two-signal model of CL activation.

Animals↗

Rapid magnetic purification of rosette-forming lymphocytes.

High gradient magnetic separation, which as previously been shown effective in extracting erythrocytes from a flowing cell suspension, has been used to separate rosetted and unrosetted human peripheral blood lymphocytes. The hemoglobin in the sheep red cells used to form rosettes was first oxidizied to the paramagnetic methemoglobin form. Samples of 50 x 10(6) lymphocytes could be processed in 10 min under sterile conditions with greater than 90% purity of the rosetted cell fraction and maintenance of T cell function in mixed lymphocyte cultures.

Animals↗