Cell Surface Immunoglobulin Research Articles

Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines.

Anti-human immunoglobulin (Ig) antibodies with single-chain specificities induced Epstein-Barr virus (EBV) in various Burkitt lymphoma lines when the corresponding Ig chain was expressed on the cell surface. The F(ab')2 fragments of IgG antibody were as potent as intact Ig, while the Fab and Fc fragments gave no induction, indicating that cross-linking of surface Ig was required for the induction. Simultaneously with EBV induction, anti-Ig inhibited the uptake of 3H-thymidine. This inhibition was also seen in EBV-genome-negative Burkitt lymphoma lines. In contrast, no effect on virus induction and cell growth was noted in lymphoblastoid lines of non-neoplastic origin. The possible relationship between cell differentiation and latency of EBV-carrier state is discussed.

42] Biosynthetic, surface labeling, and isolation of membrane immunoglobulin

Publisher Summary This chapter reviews the cell surface iodination, which is a powerful tool for the study of cell surface immunoglobulins (Ig). The surface Ig status of viable cells can be determined. Molecules which are present in low numbers or have a slow biosynthetic rate can be studied biochemically and the effect of various agents which change the surface Ig phenotype can also be monitored. The chapter explores that number of methods for cell surface labeling has been reported. The most popular is lactoperoxidase-catalyzed iodination using H 2 O 2 directly or H 2 O 2 generated by the glucose–oxidase system. If properly used, this procedure achieves the vectorial labeling of membrane but not cytoplasmic proteins and is not toxic to cells. It also discusses that the choloramine-T method utilizes a powerful oxidant followed by strong reducing conditions to terminate the reaction and is often deleterious to cells.

13] Separation of cells by affinity chromatography on protein A gels

This chapter discusses that protein A from staphylococcus aureas (SPA) reacts with the Fc region of cell-surface immunoglobulin G (IgG). The interaction between SpA and cell-surface IgG has been used to develop main techniques to isolate lymphocytes and other cells. The use of magnetic albumin microspheres containing SpA coated with IgG antibody directed against specific cell surface antigens. These microspheres adhere to cells bearing the antigen which can be removed by applying a magnet. The chapter discusses the affinity chromatography of cells on SpA-Sepharose gels. This material may be used to bind cells having IgG on their surface or cells coated with an IgG antibody specific for a particular membrane antigen. The cells are removed from the column mechanically or by elution with soluble IgG. The chapter concludes that in recent years SpA-Sepharose 6MB has become commercially available, affinity chromatography may become the method of choice for separation of IgG-bearing cells.

Phenotypic and functional characterization of murine B lymphocyte precursors isolated from fetal and adult tissues.

Murine fetal liver and adult bone marrow cells identified by monoclonal 14.8 antibody were enriched on antibody-coated polystyrene petri dishes. Cell surface immunoglobulin (sig)-bearing cells were depleted before this enrichment procedure, and the resulting preparations of 14.8+, slg- cells were characterized as to morphology, immunoglobulin gene expression, and functional potential in vivo and in vitro. All cells with detectable mu chains of IgM in the cytoplasm (cmu) were found to be included in the 14.8+ population. The enriched cells did not contain significant numbers of committed granulocyte-macrophage progenitor cells or putative hemopoietic stem cells. Selected cells from 16-day fetal liver were large, a majority of the cells had a lobulated rather than a spherical nuclear outline, and less than 1% had detectable cmu. Enriched cells from 19-day fetal liver were on the average smaller than those from 16-day-gestation liver and had a more typical lymphoid morphology; 30% were cmu+. Adult bone marrow 14.8+, slg- cells were similar to 19-day fetal liver cells in morphology, and approximately half were cmu+. These selected precursor cells retained the capacity to mature in vivo and in vitro. Fetal and adult 14.8+, slg- cells were efficient in generating newly formed B cells in vivo, and this maturation step appeared to be dependent on the presence of microenvironmental accessory cells. However, the ability of positively selected cells to mature in vitro was markedly decreased, and this potential was not rescued by providing known sources of accessory cells. Possible reasons for this difference are considered. This technique for positively selecting cells has allowed us to directly compare for the first time B cell precursors from fetal and adult tissues and will be invaluable for resolution of the cell compartments in the differentiation of B lymphocyte precursors, in the study of accessory cells known to facilitate this process, in the definition of humoral factors which may act on pre-B cells, in the study of immunoglobulin gene rearrangements which take place during normal differentiation, and for further comparative studies of fetal and adult lymphopoiesis.

The effects of interleukin 1 on human B cell activation and proliferation.

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.

Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins.

We have explored the relationship among immunoglobulin gene rearrangement, cytoplasmic immunoglobulin production, and cell surface antigen expression within 37 cases of acute lymphocytic leukemia. All 12 cases of the T cell type had germ-line kappa and lambda genes and 11 of 12 had germ-line heavy chain genes. In contrast, all 25 cases of the "non-T, non-B" classification, which lacked both definitive T cell markers and surface immunoglobulin, had rearranged immunoglobulin genes, indicating that they represent precursor cells already committed to the B cell lineage at the gene level. 14 had rearranged heavy chain genes, yet retained germ-line light chain genes, whereas 11 cases had both heavy and light chain gene reorganizations. All patterns of immunoglobulin gene rearrangement predicted by a model that proceeds from heavy chain gene recombination to light chain genes were observed. Despite the uniform presence of rearranged immunoglobulin genes, only five cases produced cytoplasmic mu-chain, one exceptional case produced gamma-chain, and another produced only lambda-chain. The cases of B cell precursor type that do not produce immunoglobulin may represent cells that frequently possess ineffectively rearranged immunoglobulin genes. Included in this group may be a set of cells trapped within the B cell precursor series because their ineffective rearrangements have eliminated certain gene subsegments necessary for the assemblage of an effective heavy chain gene. All seven cases of the non-T, non-B subgroup that bore HLA-DR but lacked CALLA (the common acute lymphocytic leukemia-associated antigen) represented the earliest recognizable stage of B cell precursors with rearranged heavy chain genes but germ-line light chain genes. Correlations here suggest that cells entering B cell development express HLA-DR and rearrange heavy chain genes before the expression of a B cell-associated antigen recognized by the antibody BA-1, the antigen CALLA, and any subsequent light chain gene rearrangements.

Determination of dissociation constants and ligand specificity of detergent solubilized surface membrane immunoglobulin a from MOPC-315

Surface membrane immunoglobulin from MOPC-315 plasmacytoma cells (smM315) was isolated by nonionic detergent lysis of radioiodinated cells and affinity chromatography on Dnp-aminohexyl-Sepharose 4B. Verification of the solubilized molecule as an integral membrane protein, distinct from secreted MOPC-315 IgA (M315) was accomplished by NaDodSO − 4-PAGE, charge-shift electrophoresis and molecular sieve gel filtration with NP-40 and deoxycholate. smM315 was compared to reduced and alkylated monomeric secreted immunoglobulins from MOPC-315, MOPC-460, and XRPC-25 by quantitative affinity chromatography (QAC) using two differently substituted Dnp-aminohexyl-Sepharose 4B resins. Unique patterns of cross-reactivity of all secreted myeloma proteins were independently established with a competitive hapten inhibition assay using 125I-Dnp 26BSA as the precipitating probe. After derivation with dinitrobenzylsulfonate, Dnp-aminohexyl-Sepharose 4B was modified with succinic anhydride which, with the inclusion of 0.03% Doc in a PBS and 0.1% NP-40 buffer, prevented nonhapten specific protein-matrix interactions during QAC. Dissociation constants determined by QAC for three ligands, (dinitrophenyl-glycine, trinitrophenyl-amino-caproate and tetramethylrhodamine) were essentially the same for smM315 and M315. Both of the other nitrophenyl binding IgA myelomas had distinct and significant differences in dissociation constants. Thus, for a differentiated antibody secreting cell which has undergone a heavy chain class switch, such as MOPC-315, the cell surface immunoglobulin has an identical ligand binding active-site as the secreted immunoglobulin.

EBV transformed B cell lines present to antigen specific T cell clones determinants different from those recognized by antibody in an HLA-DR linked restricted fashion

Human B cells nonspecifically activated by Epstein Barr virus (EBV) can present tetanus toxoid (TT) antigen to TT specific helper T cell lines and T cell clones. Presentation of TT antigen by EBV-B cells did not require the presence of TT specific B cells and did not involve B cell surface immunoglobulins. Immunosorbent purified antibody to TT added in great excess to EBV-B cells pulsed with TT for 18 hr did not inhibit the capacity of the EBV-B cells to present TT antigen. Furthermore, EBV-B cells induced proliferation in T cells in the presence of urea denatured TT which contained less than 1% TT reactive with serum anti TT antibody. Studies of TT presentation by a panel of EBB-B cells obtained from HLA typed donors indicated that T cell recognition of TT presented by EBV-B cells was MHC restricted by products of the HLA-D region some of which differed from serologically defined HLA-DR. These results indicate that the immunogenic moiety presented by EBV activated human B cells consists of antigenic determinants, which differ from those recognized by serum antibody, and of HLA-DR linked MHC determinants. This moiety is similar to that presented by monocytes. The implications of these findings for the amplification of the immune response by activated B cells are discussed.

Induction of differentiation in a case of common acute lymphoblastic leukemia.

The majority (80 per cent) of patients with acute lymphoblastic leukemia (ALL) have neither cell-surface immunoglobulin nor T-cell-associated surface antigens that would clearly identify the diseas...

Two color light scattering identifies physical differences between lymphocyte subpopulations.

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt-2 negative and Lyt-2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.

Effect of lipopolysaccharide stimulation on the transcription and translation of messenger RNA for cell surface immunoglobulin M.

Analysis of mu-specific mRNA in the B cell tumor line, BCL1, shows that the cells contain predominantly mRNA for mu chain of membrane-bound immunoglobulin M (IgM) (2.7 kb, mu m mRNA). Stimulation of the cells to Ig secretion by lipopolysaccharide (LPS) results in a 6-12 fold increase in amount of mRNA for the mu chain of secreted IgM (2.4 kb mu s mRNA). The increase in mu s mRNA is accompanied by a 3-4-fold increase in mu m mRNA. The rate of mu chain synthesis of membrane IgM in LPS-stimulated cells is, however, reduced by at least twofold, suggesting that both transcriptional and translational regulatory events are involved in the induction of B lymphocytes to secretion.

T cell enrichment and depletion of human peripheral blood mononuclear cell preparations. Unexpected findings in the study of the functional activities of the separated populations

In the present communication we present the results of a study undertaken to assess the effectiveness of several methods of depleting human peripheral blood of thymus-derived (T) cells, and to assess the purity of the T cell-enriched populations obtained. The methods of T cell depletion employed included rosetting with unmodified sheep red blood cells (SRBC) in the cold at high erythrocyte to lymphocyte ratios; rosetting with S-2-aminoethylisothiouronium bromide hydrobromide (AET)-treated SRBC; and cytotoxic depletion using anti-Leu-1, a monoclonal antibody directed toward a pan-T cell antigen, and rabbit complement. The methods used to enrich for T cells included rosetting with unmodified or AET-treated SRBC, and passage through a nylon wool column. • The purity of the T-enriched and T-depleted populations obtained was assessed by (a) repeat rosetting with unmodified SRBC, (b) staining for cell surface immunoglobulin (sIg), (c) quantitating the number of Ig secreting cells (ISC) in a reverse hemolytic plaque assay, (d) quantitating the proliferative responses to the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) and to the B cell mitogen Staphylococcus aureus Cowan (SAC), and (e) quantitating the development of ISC following culture with PWM, with or without the addition of an irradiated T cell-enriched population. • By all parameters investigated, the depletion of T cells from peripheral blood by rosetting with AET-treated SRBC or by treatment with anti-Leu-1 and complement was far more effective than by rosetting with unmodified SRBC under optimal conditions. Of particular note was the finding that functional assays (ISC at time zero (T 0), proliferative response to SAC, the development of ISC on culture with PWM) revealed a much higher degree of B cell contamination in all of the T cell-enriched populations than would have been suspected from the extent of the depletion of sIg staining cells.

Immunogenic and antigenic epitopes of immunoglobulins. II. Antigenic differences between secreted and membrane IgG demonstrated using monoclonal antibodies.

Twenty-three monoclonal antibodies with specificity for epitopes in the Fc fragment of IgG have been used to investigate antigenic differences between secreted and membrane forms of IgG produced by 2 human B lymphoblastoid cell lines (LCL). All of the monoclonals reacted with IgG secreted by the cell lines, as demonstrated by their ability to agglutinate SRBC coated with immunoglobulin isolated from culture supernatants. Membrane IgG expression was studied using direct and indirect rosette assays with antibody-coated ORBC. A surprisingly high number of antibodies, 13 on EB2 and 9 on EB4, did not bind to the cell surface immunoglobulin. These included antibodies with specificities for both C gamma 3 and C gamma 2 domain determinants. Similar results were obtained with an indirect radiobinding assay, indicating that negative results with the rosette test were not due to steric hindrance by the red cell carrier. Their performance in indirect hemagglutination indicated that most of the antibodies that did not bind to membrane IgG were of high avidity. It is concluded that the epitopes for which these antibodies are specific are not available on the cell surface. Possible explanations for the apparent antigenic differences between secreted and membrane forms of IgG are discussed against the background of previous work on the structure and mode of insertion of cell surface immunoglobulin.

Mechanism for signaling initiation and termination of B lymphocyte proliferation induced by anti-immunoglobulin.

Several lines of evidence were explored which taken together indicate that both the initiation and the termination signal for activation of rabbit lymphocytes to synthesize DNA in response to anti-rabbit immunoglobulin occurs at an immunoglobulin receptor on the surface membrane of B cells. Thus, the premature removal of anti-rabbit immunoglobulin by simply washing the cells at the 31st hour of a 48-h incubation period caused a 60% decrease in the induction of DNA synthesis. The addition of rabbit immunoglobulin to compete with B cell surface immunoglobulin for the combining sites on anti-rabbit immunoglobulin yielded a markedly diminished activation. Addition of rabbit immunoglobulin even during the latter part of a pulse label period with [3H]-thymidine was sufficient to result in reduced activation. Finally, insoluble anti-rabbit immunoglobulin at the same nominal concentration as soluble anti-rabbit immunoglobulin also was effective in inducing cells to DNA synthesis. However, it is noteworthy that under the incubation conditions used it was not possible to derive a soluble component from insoluble anti-rabbit immunoglobulin which stimulated DNA synthesis. These data have been interpreted to indicate a need for a continuous surface presence of anti-rabbit immunoglobulin to stimulate activation in a process that is not dependent upon internalization of anti-rabbit immunoglobulin.

A rapid method for quantitation of cell surface IgM by an enzyme-linked immunosorbent assay

Previous methods for quantitating cell surface immunoglobulins have been relatively tedious or have depended upon use of radioisotopes or specialized equipment (e.g., fluorescence activated cell sorter). We describe a rapid, reproducible method for measuring cell surface Ig on a population of B lymphocytes or lymphoblastoid cell lines by employing an enzyme-linked immunosorbent assay (ELISA) that uses commercially available anti-human Ig-conjugated beads (Immunobeads). Standard curves can be generated that appear to mimic the reaction kinetics of anti-μ with viable cells. Reproducibility of surface μ quantitation depends upon (1) maintaining cell viability during the reaction procedure, and (2) avoiding buffers containing diethanolamine for the alkaline phosphatase reaction. Total Ig in populations of cells can easily be estimated by reacting 10,000 Xg supernatants from lysed cells with anti-Ig beads identical to the standard curve. Combining the methods of surface and total Ig determinations, we were able to analyze quantities of membrane and cytoplasmic μ in populations of lymphoid cells.

Kappa/lambda Shifts do not occur during maturation of murine B cells.

We have used highly specific affinity-purified antibodies against mouse kappa- and lambda chains in conjunction with both immunofluorescence analysis on the FACS and enzymatic radioiodination of murine splenocytes and bone marrow cells. Our results indicate that the kappa/lambda ratio of cell surface immunoglobulin is constant during the ontogenetic development of B cells, i.e., a shift in the kappa/lambda ratio does not occur during B cell maturation. Our results suggest that the events controlling kappa and lambda expression on murine B cells are decided very early in ontogeny, before significant antigenic selection.

Tryptic and plasmic cleavage of a rat myeloma IgD

Rat cell surface immunoglobulins were analysed in sodium dodecyl sulfate polyacrylamide gels. By specific immunoprecipitation a 200,000 dalton IgM and a 165,000 dalton IgD were demonstrated. A molecule similar to the mouse and rabbit Fc receptors was non-specifically precipitated. The digestion of a rat myeloma IgD with trypsin or plasmin was studied. Although the IgD molecule was rapidly converted in Fab and Fc fragments by trypsin (1 min., 0°C) it was relatively resistant to plasmin. (Several hours at 37°C were necessary to obtain a complete conversion.) During the plasmin digestion a Facb fragment was observed. These results are discussed in regard to a possible physiological cleavage of cell surface IgD.

Immunoglobulins on the surface of hamster lymphocytes.

Cell surface immunoglobulin (Ig) is important as an antigen specific receptor on B lymphocytes. It is distributed in a clonotypic fashion and may also function in the antigen-dependent triggering of B lymphocytes. In most species, the predominant isotypes of cell surface Ig are 7–8S membrane IgM (mIgM) and IgD (mIgD). Evidence from experiments with mouse lymphoid cells indicates that the class of Ig present on the B cell can determine whether a cell is triggered or tolerized after exposure to specific antigen (reviewed in 1,2). Ontogenic development of cell surface Ig in the murine system begins with the appearance of mIgM-bearing cells and is followed by cells expressing both mIgM and mIgD. Immature murine B lymphocytes display predominantly mIgM at the cell surface and constitute a population of lymphocytes that are readily tolerized. In contrast, mature B cells express both mIgM and mIgD on their surface. Exposure of these mature cells to antigen leads to subsequent proliferation and differentiation to antibody secretion. Furthermore, removal of mIgD from the surface of these mature lymphocytes (by treatment with papain or capping by specific antisera) results in a population of cells that becomes preferentially tolerized by antigen (2). Thus, the two classes of surface Ig, mIgM and mIgD, appear to regulate the response of B lymphocytes to antigen presentation.

Immunoglobulins and immunoglobulin receptors associated with human malignant tumors.

Over the past five years 86 malignant tumors were assayed for cell surface IgG, elutable Ag-Ab complexes, and/or cell surface Fc receptors. Surface IgG was measured by indirect radioimmunoassay, using single-cell suspensions and tumor eluates. Fc receptors in tissue sections were identified by the closed chamber technique. The results confirm our previous finding that human tumors are coated in vivo with IgG and that there is a population of cells within solid tumors capable of binding Ag-Ab complexes. In addition, Ag-Ab complexes appear to attach to the cell surface membrane by the Fc zone of antibody. Ag-Ab binding was inhibited by antibody raised against the tumor. Indirect complement consumption assay indicated the presence of Ag-Ab complexes in tumor eluates. It appears that most cell surface immunoglobulin is complexed.

Cell-surface immunoglobulin and insulin receptor expression in an EBV-negative lymphoma cell line and its EBV-converted sublines.

Membrane Ig and insulin receptors were assayed in the EBV-negative Ramos lymphoma line and its EBV-converted sublines by surface fluorescence. 125I-protein A-binding assay, immunoprecipitation, and insulin receptor assay. The original Ramos line expressed surface IgM but not IgD and had a low concentration of insulin receptors. Ten of 10 converted lines expressed IgD and a variable but usually high insulin-binding capacity. Molecular weight analysis of immunoprecipitated Ig showed the presence of 2 mu-chains and 2 delta-chains. One of each heavy chain type could be characterized as membrane Ig, whereas the other was cytoplasmic. Antisera against the lambda-chain precipitated mu and delta heavy chains as well, indicating that the delta-chain expressed on the membrane of the EBV-converted Ramos lines is bound to the lambda light chain. The results show that the differentiation of a Burkitt lymphoma line is not completely "frozen" and can be induced to change in a more "activated" direction by EBV conversion.