Physiology of IgD. VII. Induction of receptors for IgD on cloned T cells by IgD and interleukin 2

The role of IgD in the immune response has remained elusive, although the predominance of IgD on the B cell surface and the paucity of IgD in serum have suggested a receptor function. In support of this hypothesis, it has recently been shown that receptors for IgD on helper T cells can be induced by exposure to IgD in vivo and in vitro. Such IgD receptor-positive T cells (i.e., T delta cells), detectable as RFC using IgD-coated SRBC, augment antibody responses. In this report, we demonstrate that cloned, antigen-specific T cells of helper phenotype show only very low percentages of IgD-RFC, if allowed to rest in vitro after antigen exposure in the absence of IL 2. Exposure to IgD or to IL 2 for 24 hr causes the IgD-specific RFC to increase as much as 25-fold to nearly 80%. Clones that have recently been stimulated with antigen, or T cell hybridomas prepared from such clones, exhibit 40 to 50% IgD-RFC before exposure and twofold higher levels after exposure to IgD. IL 2 also causes a dose-dependent induction of OgD-RFC in normal splenic T cells. Thus, antigen stimulation, IL 2 and IgD can all induce these receptors for IgD which presumably enable helper T cells to interact more effectively with IgD+ B cells.     

IgD allotypic determinants. I. Determinants expressed on murine IgD of the a allotype

Allotypes of membrane IgD of 27 strains of mice were determined with a series of anti-delta reagents, each capable of binding to the IgD of BALB/c spleen cells. One of these reagents is a newly defined allospecific rabbit anti-mouse-delta a. Typing with these reagents reveals the strain distribution of the previously described IgD.36 (Ig5.1) and IgD.43 (Ig5.4) specificities and demonstrates the existence of a new specificity, IgD.45 (Ig5.5). The results confirm the previous subdivision of the Igh-Ce haplotype into Igh-Ce, to which A mice are assigned, and Igh-Cn, to which NZB and NZW mice are assigned. In addition, it is shown that the Igh-Cd haplotype must also be subdivided. AKR mice, which express the a allele at the Igh-5(delta) locus, are designated as possessing the Igh-Cd haplotype while AL/N and C.AL20 mice, which have the e allele at the Igh-5(delta) locus, are assigned a new Igh-C haplotype designation, o.     

Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion

The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.     

Many bacterial species bind human IgD.

Forty-four bacterial strains belonging to 19 species were tested for their IgD-binding capacity by incubation with radiolabeled human IgD. A high binding of IgD to Neisseria catarrhalis and Hemophilus influenzae and a moderate binding of IgD to streptococci of the groups A, C, and G were found. Two strains of N. catarrhalis were tested for their ability to bind selectively the IgD in normal pooled serum and in three serum samples with IgD M components and were found to possess this property. Binding studies with radiolabeled IgD Fab and Fc fragments indicated that the binding mainly but not exclusively involves the CH1 region of the IgD molecule.     

Preparation of an antibody to mouse serum IgD.

Mice have recently been shown to have serum IgD. We have used affinity chromatography to partially purify mouse serum IgD, and have prepared a rabbit antiserum against this mouse Ig class. This antiserum, once adsorbed by IgD-depleted mouse serum bound to Sepharose, was isotype specific as determined by radioimmunoassay and fluorescence activated cell sorter analysis, and bound to the same splenic lymphocyte surface molecules as a hybridoma produced monoclonal anti-mouse delta antibody.     

Structure and biologic functions of human IgD. XI. Identification and ontogeny of a rat lymphocyte immunoglobulin having antigenic cross-reactivity with human IgD.

Chicken anti-human IgD antiserum (anti-delta) has demonstrated an antigenically cross-reactive homologue on rat lymphocytes. IgD and IgM are the only cell surface immunoglobulins detectable by the lactoperoxidase radiolabeling technique employed. The results indicate that, although rat surface IgD is antigenically distinct from rat IgM, the respective H chains co-electrophorese in 10% polyacrylamide-SDS gels. Rat delta-chain has an apparent m.w. of 73,000 daltons and exhibits a minor 65,000 dalton component which probably represents a partially degraded delta-chain. The ontogenic emergence of rat IgD occurs approximately 3.5 weeks after birth whereas IgM, in contrast, is apparent by 6 days of age. Thus, as in the human, IgM develops before IgD. IgD receptors are undetectable in the thymus but are present in increasing levels in spleen, blood, lymph nodes, and Peyer's patches.     

IgD receptor-mediated signal transduction in T cells

Upregulation of immunoglobulin D-specific receptors (IgD-R) on CD4+ T cells may facilitate their interaction with specific carbohydrate moieties uniquely associated with membrane IgD on B cells. Previous studies have shown that upregulation of IgD-R facilitates cognate T-B cell interactions by mediating bidirectional signaling resulting in increased antibody responses and clonal expansion of antigen-specific T cells. Murine T hybridoma cells, 7C5, constitutively express IgD-R, as has been confirmed by staining with biotinylated IgD. Earlier studies have shown that inhibitors of protein tyrosine kinase (PTK) completely prevented upregulation of IgD-R in response to oligomeric IgD, suggesting that cross-linking of IgD-R may induce signal transduction and functional consequences through one or more PTK activation pathways, leading to upregulation of IgD-R. In the present study we show that cross-linking of IgD-R by oligomeric IgD indeed results in (a) T cell activation as seen by tyrosine phosphorylation of several intracellular proteins, (b) tyrosine phosphorylation of p56 Lck and PLC-gamma in 7C5 T hybridoma cells, and (c) phosphorylation of an approximately 29-kDa band that exhibits strong affinity for IgD. We analyzed tyrosine phosphorylation of p56 Lck and PLC-gamma in BALB/c splenic T cells that were exposed to oligomeric IgD both in vivo and in vitro. In vitro cross-linking as well as in vivo followed by in vitro cross-linking of IgD-R resulted in enhanced phosphorylation of p56 Lck and moderate tyrosine phosphorylation of PLC-gamma. These results suggest that interactions between IgD-R and IgD mediate signal transduction and support our previous findings that IgD-R+ T cells enhance cognate T cell-B cell interactions and antibody production.     

Cross-reactivity of rat, mouse, and human IgD.

Highly purified sheep anti-rat lymphocyte membrane IgD (mIgD) was used to detect cross-reactivity with the putative murine-delta chain on mouse lymphocytes. Cross-reactivity is demonstrated by indirect immunofluorescent staining and by immunoprecipitation of 125I-labeled lymphocyte membrane extracts followed by electrophoresis on 10% polyacrylamide gels. In addition, cross-reactivity of anti-rat-delta with human IgD is shown by gel diffusion analysis. The anti-rat-delta reagent stained both Ig5a+ and Ig5b+ lymphocytes. Preincubation of Ig5b+ (but not Ig5a+) cells with monoclonal allotype-specific antibodies (anti-Ig5b) under capping conditions caused inhibition of staining by the sheep anti-rat-delta reagent, indicating that it is the delta-chain that is recognized on mouse lymphocytes and that the anti-rat-delta reagents does not distinguish between mouse-delta allotypes. Furthermore, absorption of the sheep anti-rat-delta serum with purified human IgD reduced subsequent staining of mouse lymphocytes by approximately 50%; staining was not affected by absorption with human IgM. This xenogeneic anti-delta antiserum appears to detect determinants on the delta-heavy chain, which are shared by at least three species of mammals, suggesting that these determinants represent important molecular features conserved during evolution.     

The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence.

Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of full-length murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an alpha-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the delta m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed.