The role of BCR isotype in B-cell development and activation.

The development and function of B lymphocytes critically depend on the non-germline B-cell antigen receptor (BCR). In addition to the diverse antigen-recognition regions, whose coding sequences are generated by the somatic DNA rearrangement, the variety of the constant domains of the Heavy Chain (HC) portion contributes to the multiplicity of the BCR types. The functions of particular classes of the HC, particularly in the context of the membrane BCR, are not completely understood. The expression of the various classes of the HC correlates with the distinct stages of B-cell development, types of B-cell subsets, and their effector functions. In this chapter, we summarize and discuss the accumulated knowledge on the role of the µ, δ, and γ HC isotypes of the conventional and precursor BCR in B-cell differentiation, selection, and engagement with (auto)antigens.

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials.

cDNAs encoding IgM heavy chain constant region (Cmu) were isolated from two metatherians (marsupials)--the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cmu in both species. The domain size and structure of the marsupial Cmu sequences were compared with other Cmu sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.

Cloning and structural analysis of IgM (mu chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica.

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (gamma chain), IgE (epsilon chain) and IgA (alpha chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (mu chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.

Balanced efficiencies of splicing and cleavage-polyadenylation are required for mu-s and mu-m mRNA regulation.

The relative abundance of the RNAs encoding the membrane (mu-m) and secreted (mu-s) forms of immunoglobulin mu heavy chain is regulated during B cell maturation by a change in the mode of RNA processing. This regulation depends on a competition between two mutually exclusive RNA processing reactions, cleavage-polyadenylation at the microseconds poly(A) site and splicing of the Cmu4 and M1 exons. Previously, the efficiencies of these two reactions were altered independently. When an efficient processing signal replaced the normal suboptimal signals of the mu gene, a single RNA product was produced exclusively. In this report, two efficient signals are combined in a single mu transcript and shown to restore a processing balance such that two mRNAs can once again be alternatively processed from a single RNA precursor. The ratio of the two RNAs generated from these mu genes containing balanced competing strong splice and cleavage-polyadenylation reactions display the expected developmental shift when expressed in B cells and plasma cells. Therefore, the balance between cleavage-polyadenylation and splicing efficiencies is critical to the developmentally regulated expression of mu-s and mu-m mRNA. Also shown here is that the entire mu-m region, including the M1 and M2 exons and the mu-m poly(A) site, can be replaced with SV40 splice and poly(A) sequences. Regulation is maintained in these mu genes, indicating that no specific sequences within the mu-m region are required.

Production of monoclonal antibodies with haemagglutination-inhibition activity to the Skalica strain from the tick-borne encephalitis complex.

Hybridomas secreting monoclonal antibodies with haemagglutination-inhibition (HI) activity to the Skalica strain of tick-borne encephalitis (TBE) complex were prepared by the fusion of P3-NS1-Ag4-1 myeloma cell line with spleen cells of BALB/c mice immunized with the purified Skalica strain. The highest titres of monoclonal antibodies obtained from the hybridomas S-9, S-15 and S-16 ranged from 512 to 10,240, respectively; the ascitic fluid contained as many as 4.6 mg/ml of monoclonal antibodies. Its analysis by Ouchterlony's double immunodiffusion, agarose electrophoresis, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of monoclonal antibodies with mu isotype of the heavy and kappa isotype of the light chain. The specificity of the monoclonal antibodies was proved using 11 different antigens from family Togaviridae in the HI test.

Control of IgM synthesis in the murine pre-B cell line, 70Z/3'.

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.

Characterization of multiple immunoglobulin mu-chains synthesized by two clones of a B cell lymphoma.

We have identified 3 species of mu-chain synthesized by mouse WEHI 279 lymphoma cells that differ in cellular location, size and charge--mui (internal), mum (membrane), and mus (secreted). Lactoperoxidase and galactose oxidase labeling experiments localize the mum chain to the plasma membrane and the mui chain to the cytoplasm. Pulse-chase experiments demonstrate that the mui pool contains precursors to both mum and mus chains. Comparative peptide mapping studies and cell labeling in the presence of tunicamycin suggest that the mum chain is 2000 daltons larger than the mus chains and that the difference is due to polypeptide alterations rather than carbohydrate differences. The WEHI 279 lymphoma has differentiated while in culture to a more advanced stage in the pathway of B cell differentiation.

Chicken high molecular weight immunoglobulin (IgM) allotypes: localization on the heavy chains and proposed nomenclature.

An alloantiserum recognizing a genetic marker (CM-1.4) on chicken 17S Ig was generated by immunizing jungle fowl with Salmonella O-anti-Salmonella O (UCD 3) agglutinates. Specificities CM-1.4 and M1a were present on 7S subunits and H chains based on their ability to inhibit in RIA the binding of alloantibody (anti-CM-1.4 or anti-M1a) to UCD 3 125I-17S Ig. CM-1.4 appears to be fully expressed on 7S subunits but is altered on H chains. In contrast, anti-M1a bound both 7S subunits and H chains with decreased avidity indicating that the M1a specificity was altered in both preparations. Since CM-1.4 and M1a are present on 17S Ig H chains, and absent from 7S Ig, they probably represent allotypic markers in the constant region of 17S Ig H chains. Having assigned two 17S Ig allotypic specificities to H chains, we propose that the locus controlling their expression be called CM-1. Based on the distribution of the CM-1.4 and M1a allotypes in inbred lines of chickens, 3 alleles are defined at the CM-1 locus.