IgD-like Molecules Research Articles

Identification of a dog IgD-like molecule by a monoclonal antibody

IgD has not been identified in dogs. We produced a monoclonal antibody (mAb) designated 9B during the production of hybridomas to dog IgE. Using Western blot analysis under non-reducing conditions, the mAb (9B) recognized a predominant protein band of 185 kDa which was also recognized by anti-dog IgG F(ab′) 2, suggesting that this 185 kDa protein is an immunoglobulin (Ig) containing light chains. Under reducing conditions, the mAb (9B) recognized only one protein band of 55 kDa which presented a distinct molecular weight (MW) and immunoreactivity from the dog τ, μ, α, and ϵ chains. The 55 kDa band did not react with anti-dog IgE, IgM, IgA, and IgG, but did react with the mAb (9B). The MW was 75 kDa for the ϵ chain, 77.5 kDa for the μ chain, 58 kDa for the α chain, and 52 kDa for the τ chain. Further, by immunofluorescent staining, this Ig recognized by the mAb (9B) was found on the surface of dog lymphocytes. Studies of this dog Ig with the mAb revealed that this Ig bound to protein A and protein G-Sepharose, and that its enzyme-linked immunosorbent assay (ELISA) activity as measured by the mAb (9B) did not change after heating at 56°C for 2 h. Ragweed-specific IgG, IgE, and this newly defined Ig significantly increased when dogs were immunized with ragweed extract. These data suggest that this Ig is a previously unrecognized IgD-like molecule in dogs.

The appearance of an IgD-like molecule on pig lymphocytes during ontogeny.

An IgD-like molecule was found on the surface of pig fetal lymphocytes using guinea pig anti-human IgD antisera and indirect immunofluorescence. The binding of antiIgD antisera could be specifically inhibited with human IgD. Surface IgD-like molecules were found later in ontogeny than surface IgM (after about 70 d of gestation in the spleen). There is evidence for their endogenous origin. The number of lymphocytes bearing IgD-like molecules rapidly increased during ontogeny.

Cross-reactivity of human and a putative pig IgD. Pig IgD-like molecules as serum and lymphocyte components.

Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, and susceptibility of the heavy chains, light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.

Demonstration of A Rabbit Cell Surface Ig That Bears Light Chain and VH, But Lacks µ-, α-, and γ-Allotypes-Rabbit IgD?

Abstract Based on previous biochemical studies of rabbit sIg, we postulated that if rabbits possessed an IgD-like molecule, its heavy chain might co-electrophorese with the µ-heavy chain on reduced SDS-PAGE. Accordingly, we studied Ig from rabbit spleen cells radiolabeled biosynthetically or by cell surface iodination. Cell lysates were prepared and immunoprecipitation studies were performed by standard techniques. Sequential absorptions in which total removal of µ-allotype (n-locus) was followed by immunoprecipitation with anti-a(VH) or anti-b (kappa) showed residual band(s) with µ-relative mobility on SDS-PAGE under reducing conditions. Similar immunoprecipitations with anti-gamma (γ) or anti-alpha (α) reagents failed to precipitate the residual band with µ-mobility. The residual band was found in both radioiodinated and biosynthetically radiolabeled lysates. When direct immunoprecipitation was carried out in the absence of proteolytic enzyme inhibitors, the SDS-PAGE pattern of total Ig under reducing conditions frequently showed three peaks, corresponding to µ (MW ∼70,000), light chain (MW ∼23,000), and an unidentified heavy chain (MW ∼65,000). The second heavy chain peak was not immunoprecipitated with µ, γ, or α allotype sera. Thus, we have identified a “new Ig” in the rabbit that bears VH and light chain, and lacks µ, γ and α allotype markers. The molecule appears to be proteolytically labile, but has an undegraded heavy chain that co-electrophoreses with µ-chains on reduced SDS-PAGE. The similarities to published data on human and rat lymphocyte IgD strongly suggest, but do not formally prove, that we have identified the rabbit homologue of IgD.

Introduction -- the immunoglobulin-like T cell receptor problem.

No problem in modern immunology has been more frustrating and controversial than that of the nature of the receptor for antigen on T lymphocytes. Since T cells, like antibodies and B cells, exhibit exquisite specificity for antigen, it is reasonable to propose as Ehrlich first did more than 80 years ago that the cell surface antigen receptor must be antibody (1). A good deal of experimental support was obtained for this concept in the late 60’s and early 70’s (reviewed in 2), but the hypothesis was not universally accepted because evidence from various approaches indicated that the T cell receptor, if an immunoglobulin (Ig), could not be identical to the known serum isotypes (3,4). However, a precedent for Igs associated only with the plasma membrane was set by the discovery of the IgD-like molecule of murine B cells (3,5–7) which does not occur in serum. It is not surprising that surface Ig molecules, which exist in a hydrophobic environment, differ from their serum counterparts, which exist in an aqueous miliue.

Antigen recognition by T lymphocytes

Data of recent studies based upon three approaches to the problem of the T cell receptor for antigen, namely, use of ‘anti-receptor’ (≡ anti-idiotype) antibodies, utilization of antisera made against circulating antibodies in association with physicochemical characterization procedures, and phylogenetic investigations, support the conclusion that this recognition molecule is an immunoglobulin, but that it represents an isotype distinct from usual circulating antibodies and B cell surface IgM- and IgD-like molecules.

Hapten-specific B Lymphocytes: Enrichment, Cloning, Receptor Analysis, and Tolerance Induction

In this paper, we review experiments verifying clonal selection through the use of hapten-fractionated B lymphocytes of such uniformity that up to one cell in three can be induced to form an anti-hapten antibody-forming clone in vitro; the use of such cells in studies on antigen-induced receptor aggregation and modulation using immunogenic or tolerogenic antigen concentrations; application to the tolerance problem of the cloning methods devised for the study of these cells; studies of the IgM- and IgD-like receptors of hapten-specific cells; and the use of an anti-delta allotype serum to study the distribution and function of IgD-like molecules on the B-lymphocyte surface.

Cell Surface Immunoglobulin

Abstract 125I-membrane IgM, 125I-membrane IgD-like molecules, and their constituent chains from iodinated murine splenocytes were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent m.w. of the heavy chains decreased as the acrylamide concentration was raised. The membrane µ-chain had a slower mobility than did µ-chain from secreted IgM. Unreduced IgM and IgD-like molecules had mobilities consistent with an H2L2 structure. Intact IgD-like molecules were replaced after overnight dialysis by molecules with the properties of HL. Unreduced surface IgM had a slower mobility than that of monomeric IgM obtained by partial reduction of secreted IgM.

Continuous growth of mitogen-reactive B lymphocytes.

B-cell mitogens induce continous growth of murine lymphocytes in suspension cultures. Continuous growth is observed only at low cell densitiess, below 10(6) cells/ml, in regularly renewed medium containing beta-mercaptoethanol and a growth-supporting fetal calf serum. Continuous growth of B lymphocytes in culture appears to be favored by conditions under which maturation of Ig-secreting plaque-forming cells does not occur. On the other hand, maturation is observed whenever stimulated B cells cease to divide. This maturation leads to IgM secretion followed by secretion of IgG- or IgD-like molecules. Neither continuous growth of B cells nor the 'switch' to secretion of IgG- or IgD-like molecules requires the presence of T cells.

Cell Surface Immunoglobulin. XIII. Distribution of IgM and IgD-Like Molecules on Small and Large Cells of Mouse Spleen

The distribution among murine spleen cells of a newly described class of surface immunoglobulin (Ig) with properties similar to human IgD was studied. Splenocytes were separated on the basis of size and the surface Ig on large cells (sedimenting faster than 6 mm/hr in a 1 times G velocity gradient) and small cells (sedimenting between 2.5 and 3.0 mm/hr) was analyzed. Spleen cells from young animals had virtually only IgM on the large cells but had substantial amounts of IgM and the IgD-like molecule (IgD) on small cells. Spleen cells from older animals, which have larger amounts of IgD, had IgM and IgD on both cell types; however, the amount of IgD relative to IgM on the large cells was always substantially less than that on the small ones. These observations taken together with those of other investigators support the hypothesis that a large lymphocyte with surface IgM is the precursor of a small lymphocyte with both surface IgM and IgD.

Cell surface immunoglobulin. XI. The appearance of an IgD-like molecule on murine lymphoid cells during ontogeny.

An Ig molecule containing L chains and H chains similar to human delta-chains has been detected on the surface of radioiodinated murine lymphoid cells. Newborn mice have only IgM on their splenocytes. Between 10 and 15 days, the IgD-like molecule appears and increases in amount until 3 mo of age, when it is the predominant cell surface Ig in terms of radioactivity. IgD is found only in peripheral lymphoid tissues and is present in larger amounts on peripheral lymph node cells (approximately 85% of surface Ig) than on splenocytes (approximately 50%). IgD is also present in comparable amounts on cells from both nu/nu and germfree mice, indicating that its expression may be independent of both thymic influence and antigenic stimulation. These studies suggest that there is a switch from cell surface IgM to IgD that occurs during differentiation of virgin B lymphocytes in the spleen.