Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation (original) (raw)
Related papers
Journal of Experimental Medicine, 1988
The perturbation of membrane immunoglobulin (mIg)' on B lymphocytes can have diverse effects on B cell physiology. Immature B cells and certain activated B cell populations are particularly susceptible to inhibition (tolerance) after mIg crosslinking (1-4), while mature, resting B cells are characteristically stimulated to DNA synthesis (4-6) . Because both stimulatory and inhibitory signal transduction appear to involve similar early biochemical reactions (1, 6), it is of some interest that the ligand dose requirements for achieving these distinct functional phenomena have generally been found to differ by one or more orders of magnitude (3, 7-10) . This suggests that the ligand binding requisites for triggering B cell tolerance may be significantly different from those for directly triggering B cell clonal expansion .
Molecular Analysis of Ligand Binding Requisites for Human B Cell Clonal Expansion and Tolerance
2000
The perturbation of membrane immunoglobulin (mIg)' on B lymphocytes can have diverse effects on B cell physiology. Immature B cells and certain activated B cell populations are particularly susceptible to inhibition (tolerance) after mIg crosslinking (1-4), while mature, resting B cells are characteristically stimulated to DNA synthesis (4-6). Because both stimulatory and inhibitory signal transduction appear to involve similar early biochemical reactions (1, 6), it is of some interest that the ligand dose requirements for achieving these distinct functional phenomena have generally been found to differ by one or more orders of magnitude (3, 7-10). This suggests that the ligand binding requisites for triggering B cell tolerance may be significantly different from those for directly triggering B cell clonal expansion. In this report, we have attempted to rigorously evaluate the binding requisites for eliciting the activation, or alternatively, inactivation of B lymphocyte DNA synthesis through mIg. With the use of a large panel of anti-human IgM mAbs, we examine how the site specificity, the affinity, and the valency of epitopes bound on mIgM affect the capacity for ligand-induced regulation of DNA synthesis in the appropriately sensitive human B lymphocytes. The data demonstrate that the IgM binding requisites for inducing inhibition of B cell DNA synthesis significantly differ from those for stimulation. These studies may be revelant to the control of human B cell clonal expansion by antiidiotype antibodies and rheumatoid factors.
Molecular Immunology, 1987
The domain binding specificity of 19 murine anti-human IgM monoclonal antibodies (MoAbs) that have shown considerable heterogeneity in the transduction of stimulatory and inhibitory signals to B lymphocytes was evaluated by competition radioimmunoassays. Through the use of: (i) enzymatic fragments of IgM which each encompass more than a single C, domain, i.e. Fc,~ and F(ab'),p, (ii) isolated single domains, Cpc,, Cp,, and Cpq, and (iii) mu heavy chain disease proteins, nine anti-IgM MoAbs were found to have Cp, domain specificity, five to have Cpc, specificity, and five others to have Q4 specificity. Ineffective binding to isolated p chain demonstrated that Cp,-specific MoAbs were directed to epitopes which require light chain for expression. The lack of binding of the Cp,-specific MoAbs to CNBr cleavage fragments of Fc,~ suggest that the determinants recognized by these MoAbs may also be conformational in nature. Cross-inhibition analyses were used to determine the number of unique epitopes recognized by the anti-IgM MoAbs. Results from these experiments showed that: (i) eight of the nine MoAbs specific for Cp, likely bind to a single epitope, or very proximate epitopes, (ii) the five Q-specific MoAbs recognize at least three distinct epitopes, and (iii) the five Cp,-specific MoAbs each recognize a separate determinant. A comparison of the known B cell activating properties of these MoAbs with their specificitv for the various segments of the IgM molecule indicate that mitogenicity cannot be attributed to selective binding to any one domain.
Up-regulation of the MHC class II molecules on B cells by peptide ligands
Journal of immunology (Baltimore, Md. : 1950), 1994
MHC class I and class II molecules present peptide Ag to T lymphocytes. Peptides are critical in class I heavy chain folding and/or stable association with beta 2m. A recent study suggests the role of peptide Ag binding for MHC class II alpha- and beta-chain heterodimers to enter into a compact state and allow their transport to the cell surface. We have investigated the effect of peptide ligands on the expression of MHC class II I-A(d) molecules on the B cell hybridoma, TA3. These cells, when cultured in vitro, gradually lost the surface expression of I-A(d) molecules. Incubation with peptides, having high affinity for binding to intact I-A(d) molecules, significantly increased the surface expression of I-A(d) in less than 24 h. The ability of peptides to induce increased expression of I-A(d) correlated with the affinity of peptide to intact I-A(d), and an I-Ak-restricted peptide did not have an effect on I-A(d) expression. The effect could be reversed after the removal of the pept...
The Journal of Immunology, 2010
Membrane-bound IgE (mIgE) is part of the IgE-BCR and is essential for generating isotype-specific IgE responses. On mIgE + B cells, the membrane-bound «-chain (m«) exists predominantly in the long isoform, m« L , containing an extra 52 aa C«mX domain between CH4 and the C-terminal membrane-anchoring segment; the short isoform of m«, m« S , exists in minor proportions. C«mX thus provides an attractive site for immunologic targeting of mIgE + B cells. In this study, we show that nine newly prepared C«mX-specific mAbs, as well as the previously reported a20, bound to mIgE.Fc L-expressing CHO cells, while only 4B12 and 26H2 bound to mIgE.Fc L-expressing B cell line Ramos cells. The mAb 4B12 bound to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of C«mX. Expression of Iga and Igb on the mIgE.Fc L-CHO cells reduces the binding of a20 to C«mX as compared with that of 4B12 and 26H2. The chimeric mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, lysed mIgE.Fc L-Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 demonstrated Ab-dependent cellular cytotoxicity toward mIgE.Fc L-Ramos cells in a dose-dependent fashion. In cultures of PBMCs from atopic dermatitis patients, c4B12 and c26H2 inhibited the synthesis of IgE driven by anti-CD40 and IL-4. These results suggest that 4B12 and 26H2 and an immunogen using the peptide segments recognized by these mAbs are potentially useful for targeting mIgE + B cells to control IgE production.
Affinity maturation of secreted IgM pentamers on B cells
International Immunology, 2004
We prepared a series of hapten-BSA conjugates with varying ratios of biotin to measure ligand± receptor interactions on B cells by¯ow cytometry using avidin for detection. Surface plasmon resonance measurements of the interaction with a monoclonal anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody suggested that NP 5-BSA or NP 7-BSA harboring 29 or 23 biotin molecules (NP 5-BSAbio 29 or NP 7-BSA-bio 23) would be suitably sensitive for¯ow cytometric analysis. By using NP-BSAbio, we analyzed NP-binding cells in immunized mice. Unexpectedly, 30±40% of spleen cells expressing IgM could bind to NP 5-BSA or NP 7-BSA after immunization of mice with NP 40-chicken g-globulin. The proteins binding to NP 7-BSA-bio 23 on the cell surface were analyzed by immunoprecipitation and western blotting. Surprisingly, most of the proteins binding NP-BSA-bio on the cell surface were not the membrane form of IgM monomer, but a secreted IgM pentamer. It is likely that the IgM pentamer bound through Fc receptors for polymeric IgA or IgM and contributed to antigen binding. Comparison of the binding ratio of NP 0.9-BSA:NP 5-BSA between B cells of primary and secondary immunization suggested that the af®nity of IgM matured during immunization.
The Journal of Immunology, 2004
Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H 2 L 2 Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties.
Molecular Basis of B-Cell Activation
Scandinavian Journal of Immunology, 1974
The present experiments were performed in an attempt to investigate the nature of the surface receptor on B lymphocytes responsible for triggering of these cells. B-cell mitogenicity of unsubstituted dextrans, quantitated by activation of DNA and antibody synthesis was detected only if the ligand had a MW higher than 7 X IO*. Above this threshold mitogt-nicity increased linearly with the log of the MW. Substitution of the polymeric structure with lipid residues did not result in increased mitogenicity of the conjugate. However, sulphate substitution of the sugar units greatly enhanced the ability of the conjugates to activate DNA synthesis and, to a much smaller extent, antibody synthesis. Mitogenicity of sulphate derivatives was independent of their MW. Another polyanionic derivative (carboxymethy!) did not show increased mitogenicity. whereas a low-MW compound very similar to dextran sulphate (pentosan sulphate) was highly active. The activation induced by dextrans was immunologically nonspecific and caused induction of polyclonal antibody synthesis. The activated cells presumably belong to a subset Q{ B cells at a rather premature stage of differentiation. These findings suggest that the mitogenic signal is delivered to the cells by single sites at the membrane. These sites appear to have the capacity to interact with polysaccharide structures or releated conformations. The polymeric structure of the active ligands is not a necessary requirement for mitogenicity and seems to have the accessory function of providing multipoint binding to low-affinity receptors.