Characterization of the Cell Surface Phenotype and Regulatory Activity of B-Cell IgD Low (BDL) Regulatory B Cells.

B-cell IgD Low (BDL) B cells have been shown to promote immunological tolerance by inducing proliferation of CD4+Foxp3+ T-regulatory cells (Treg) in a glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL, Tnfsf18)-dependent manner. BDL cells constitute a small subset of splenic B lymphocytes that, in mice, are characterized by the B220+IgMintCD21intCD23+CD93-IgDlow/- cell surface expression profile. In this chapter, we show the flow cytometry gating strategy developed to identify and purify BDL. In addition, we describe an in vitro assay and two in vivo assays to assess BDL regulatory activity by quantitating Treg expansion/proliferation and indicate how they can be used in mouse models of disease. Collectively, these methods are useful to track and quantitate BDL and Treg numbers and assess their regulatory activity in inflammatory disease models.

Proteomic Characterization of Immunoglobulin Content in Dermal Interstitial Fluid.

Microneedles have been demonstrated to be a minimally invasive technique for sampling dermal interstitial fluid (ISF). Shotgun quantitative proteomics has already identified hundreds of proteins in ISF and quantitatively compared the proteome to matching serum and plasma. Interstitial fluid was determined to be a viable minimally invasive alternative to blood-derived fluids. In this communication, we re-examined the proteomic data from previous work to determine the diversity of immunoglobulins present compared with serum and plasma. Similar to our previous findings regarding the proteomic content across fluid types, ISF had a similar composition of IgG, IgA, IgD, and IgE antibodies as plasma or serum and lower quantities of IgM, which reflects the relative concentrations of dermal tissue T-cell and B-cell populations, indicating that the Ig's were likely locally derived. This work has significant implications for the utility of measuring Ig's in ISF for the clinical diagnosis of immunological diseases and skin infections. Data are available via ProteomeXchange with identifier PXD012658.

Isolation of murine and human immunoglobulin m and murine immunoglobulin D.

This unit describes two classical protocols for the purification of IgM-dialysis of ascites fluid, tissue culture medium, or bioreactor supernatants against distilled water to precipitate pure IgM, and ammonium sulfate precipitation. Both protocols can be followed by size-exclusion chromatography to obtain a highly purified product. Recently, an affinity method for purification of IgM has been developed using mannan-binding protein, and is described here. The third approach presented is a one-step IgD purification method, designed specifically for murine derived samples, that uses Sepharose coupled to lectin derived from the seeds of Griffonia simplicifolia-1. This represents a simple, rapid, and gentle, approach to isolating this highly labile immunoglobulin from IgD-containing ascites or hybridoma sources.

Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD.

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

Purification of IgD from human serum--a novel application of recombinant M. catarrhalis IgD-binding protein (MID).

Moraxella catarrhalis IgD-binding protein (MID) is a multimeric outer membrane protein belonging to the family of autotransporters. The IgD-binding domain of MID is located between amino acids MID 962-1200 and binds to amino acids 198-224 of the IgD C(H)1 region. In the present study, we describe a method to purify IgD from serum with high levels of IgD using a two-step affinity chromatography process. The first step involves depletion of MID-specific antibodies of all classes from serum using the non-IgD-binding fragment MID(1000-1200). This step is followed by selective capture of IgD with MID(962-1200). Furthermore, we demonstrate that the eluted IgD is pure, intact and functional for use in downstream applications. Our approach reduces the non-specificity commonly associated with lectin-based IgD purification regimes that rely on glycosylation of the IgD molecule.

Semi-extended solution structure of human myeloma immunoglobulin D determined by constrained X-ray scattering.

Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R(G) of 6.9(+/-0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R(G) of 6.1-6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars.

Artiodactyl IgD: the missing link.

IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig delta genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cdelta gene appears to have been duplicated from the Cmu gene >300 million yr ago. The ruminant mu CH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, approximately 20 million yr ago, inserted upstream of the delta gene hinge regions, and later modified by gene conversion. A short Sdelta (switch delta) sequence resulting from the second duplication, is located immediately upstream of the bovine Cdelta gene and directs regular mu-delta class switch recombination in the cow. The presence of Cdelta genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage.

The IgH locus of the channel catfish, Ictalurus punctatus, contains multiple constant region gene sequences: different genes encode heavy chains of membrane and secreted IgD.

The delta-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to C micro 1, seven C domain-encoding exons (delta1-delta7), and a transmembrane tail. The presence of cDNA forms showing splicing of delta7 to an exon encoding a secretory tail was interpreted to indicate that membrane (deltam) and secreted (deltas) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three delta C region genes, each linked to a micro gene or pseudogene. The first (IGHD1) is located 1.6 kb 3' of the functional C micro (IGHM1). The second (IGHD3) is positioned immediately downstream of a pseudo C micro (IGHM3P), approximately 725 kb 5' of IGHM1. These two delta genes are highly similar in sequence and each contains a tandem duplication of delta2-delta3-delta4. However, IGHD1 has a terminal exon encoding the transmembrane region, whereas IGHD3 has a single terminal exon encoding a secreted tail. The occurrence of IGHD3 immediately downstream of a micro pseudogene indicates that the putative deltas product may not be expressed as a chimeric micro delta molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish deltam transcripts appear to originate from IGHD1, whereas deltas transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed delta gene. The third delta (IGHD2) is associated with a pseudo C micro (IGHM2P); its presence is inferred by Southern blot analyses.

Purification of immunoglobulin M and immunoglobulin D.

This unit describes two classical protocols for the purification of IgM - dialysis of ascites fluid, tissue culture medium, or bioreactor supernatants against distilled water to precipitate pure IgM, and ammonium sulfate precipitation. Both protocols can be followed by size-exclusion chromatography to obtain a highly purified product. Recently an affinity method for purification of IgM has been developed using mannan binding protein, and is described here. The third approach presented is a one-step IgD purification method, designed specifically for murine derived samples, that uses Sepharose coupled to lectin derived from the seeds of Griffonia simplicifolia-1. This represents a simple, rapid, and gentle approach to isolating this highly labile immunoglobulin from IgD-containing ascites or hybridoma sources.

Immunoglobulin D (IgD) of Atlantic cod has a unique structure.

A new immunoglobulin heavy-chain gene with some homology to mammalian IgD was recently cloned from the channel catfish and Atlantic salmon, two species of teleost fish. We have cloned and sequenced a new H-chain gene from Atlantic cod (Gadus morhua L.) which has clear similarities to these genes, but which also differs in several ways. The similarities of catfish, salmon, and cod delta to the mammalian delta genes are sequence homology, location immediately downstream of IgM (mu), and expression by alternative splicing rather than class switching. A unique feature of catfish, salmon, and cod delta is the chimeric nature of the gene product, as the mu1 exon is spliced to the delta1 exon. Several unique features of cod IgD were found: (1) a deletion of the delta3, delta4, delta5, and delta6 domains described in catfish and salmon IgD, (2) a tandem duplication of a part of the delta locus including the delta1 and delta2 domains, (3) the presence of a truncated delta7 domain downstream of the deltaTM exons, and (4) the separation of the duplicated domains by a short exon (deltay) which has homology to a conserved part of the transmembrane exon 1 (TM1) of some H-chain isotypes. This unique organization of the delta locus of cod probably developed after the evolutionary split from the catfish and salmon branches.

Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells in vitro.

Activation of resting B cells requires an initial triggering of the B cell antigen receptor (BCR) and secondary stimuli through various cytokine receptors and B cell activation molecules including CD40. We found that activation of B cells through CD40 is selectively inhibited by an immunosuppressant drug, rapamycin. This effect of rapamycin on anti-CD40-mediated activation of B cells was observed using three different in vitro assays. Rapamycin suppressed the anti-CD40-induced proliferation of splenic B cells, suppressed differentiation to surface IgMhigh/IgDlow B cells, and inhibited an anti-CD40-mediated prevention of apoptosis induced by BCR cross-linkage of WEHI-231 cells. We next examined several known CD40 signal transduction pathways to identify the target of rapamycin in stimulated B cells. Rapamycin did not inhibit the activation of c-Jun N-terminal kinases (JNKs) induced by anti-CD40 stimulation nor the activation of immediate nuclear transcription factors of NF-kappaB. Therefore, rapamycin affects a novel element of the CD40 signal transduction pathway which influences the proliferation, differentiation, and prevention of apoptosis of B cells.

Identification of Toxoplasma gondii antigens involved in the IgM and IgG indirect hemagglutination tests for the diagnosis of toxoplasmosis.

Crude Toxoplasma gondii antigens represent raw material used to prepare reagents to be employed in different serologic tests for the diagnosis of toxoplasmosis, including the IgM and IgG indirect hemagglutination (IgG-HA and IgM-HA) tests. So far, the actual antigenic molecules of the parasite involved in the interaction with agglutinating anti-T. gondii antibodies in these tests are unknown. The absorption process of serum samples from toxoplasmosis patients with the IgG-HA reagent (G-toxo-HA) demonstrated that red cells from this reagent were coated with T. gondii antigens with Mr of 39, 35, 30, 27, 22 and 14 kDa. The immune-absorption process with the IgM-HA reagent (M-toxo-HA), in turn, provided antibody eluates which recognized antigenic bands of the parasite corresponding to Mr of 54, 35 and 30 kDa, implying that these antigens are coating red cells from this reagent. The identification of most relevant antigens for each type of HA reagent seems to be useful for the inspection of the raw antigenic material, as well as of reagent batches routinely produced. Moreover the present findings can be used to modify these reagents in order to improve the performance of HA tests for the diagnosis of toxoplasmosis.

Role of two-dimensional electrophoretic analysis in the diagnosis and characterization of IgD monoclonal gammopathy.

Over a period of 5 years, an isolated light chain (kappa = 9, lambda = 12) was detected in 21 sera by immunofixation electrophoresis. Further analysis with anti-delta- and anti-epsilon-specific antisera identified four delta heavy chains, all associated with a lambda light chain, and no epsilon heavy chains. For evaluation of the role of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in the diagnosis of IgD paraproteins, as a possible alternative or complement to immunofixation, IgD paraproteins were retrospectively analyzed by 2D-PAGE. Delta Heavy chains migrated to gel areas clearly distinguishable from other heavy chains alpha, gamma, or mu, and in a wide range of isoelectric points (pI: 5.4-8). In one serum, the monoclonal delta chain had a pI range comparable to that of albumin and was undetectable. However, all four delta chains were easily identified when analyzed from affinity-purified immunoglobulin fractions. These observations showed the following: 1) IgD paraproteins are not rare among apparently isolated monoclonal light chains detected by routine immunofixation, strongly confirming the need for further analysis with anti-delta antisera, before assumption of a light-chain disease. 2) 2D-PAGE analysis of affinity-purified immunoglobulin fractions allowed correct identification of IgD monoclonal gammopathies in all cases. 3) However, although 2D-PAGE analysis is now easy to perform, well standardized, and highly sensitive, this technique remains time-consuming and expensive, and does not appear suitable for routine practice as a first-line diagnostic procedure. 2D-PAGE should find its place as a complement to immunofixation and in the definitive demonstration, in selected ambiguous cases, of the clonal pattern of a suspected gammopathy at immunofixation.

A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD.

The alpha-D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95-99% pure as determined by SDS-PAGE and represents 90-95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.

Differences in glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially enable transduction of different signals.

Studies presented here demonstrate that IgM and IgD molecules on normal murine B lymphocytes exist in different, noncovalently associated molecular complexes containing distinct but potentially related glycoproteins. The glycoproteins in these complexes, particularly those associated with IgD, show striking differences in various lymphoid organs and in X-linked immunodeficient (Xid) mice. These differences are due in part to post-translational processing. They apparently reflect the differential expression of the Ig-associated glycoproteins in the various B cell subpopulations and lineages and the differential distribution of the subpopulations and lineages in the various lymphoid organs. In addition, they reflect structural differences in the IgM and IgD complexes which, we suggest, permit differential signal transduction by IgM and IgD on the same B cell.

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM.

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.

Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.

The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells.

Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both allotypes.

The lectin jacalin from jackfruit seeds shows a human IgA-subclass specificity by gel precipitation and Western blotting. However, its reactivity with IgA2 is a matter of controversy. We further studied the immunoglobulin isotype specificity of jacalin by affinity chromatography with myeloma sera and by inhibition of jacalin binding to solid-phase IgA1 by purified monoclonal immunoglobulins. The lectin proved to bind IgA2 of both allotypes with a lower apparent affinity than for IgA1 and IgD.

Interaction between human IgD and ricinus agglutinin.

The results show that monoclonal IgD reacts specifically with ricinus agglutinin (molecular weight 120,000) (RcAI). The reaction between IgD and RcAI was inhibited by galactose and lactose but not by glucose, indicating that the interaction is mediated by galactose containing carbohydrate units located in or near the sigma-hinge region. Affinity chromatography on RcAI-Sepharose 4B was successfully used to isolate monoclonal IgD from the IgD myeloma plasma.