Somatic immunoglobulin hypermutation

Immunoglobulin hypermutation provides the structural correlate for the affinity maturation of the antibody response. Characteristic modalities of this mechanism include a preponderance of point-mutations with prevalence of transitions over transversions, and the mutational hotspot RGYW sequence. Recent evidence suggests a mechanism whereby DNA-breaks induce error-prone DNA synthesis in immunoglobulin V(D)J regions by error-prone DNA polymerases. The nature of the targeting mechanism and the trans-factors effecting such breaks and their repair remain to be determined.     

Somatic hypermutation in antoimmune thyroid disease

Summary: Autoimmune thyroid disease is one of the most common autoimmune diseases. There is typically patient antibody (Ab) reactivity to one or more of the antigens thyroglobulin (Tg), thyroid peroxidase (TPO) and the thyroid stimulating hormone receptor (TSFlr). With the advent of combinatorial library technology, there has been an enormous increase in the number of sequences from Ab to Tg and TPO, The repertoire of both Tg and TPO Ab is restricted and indicates the importance of somatic hypermutation in the development of the high affinity, Ab response. However, there are still too few sequences to determine patterns in which the mutation occurs, which residues are introduced during substitution and how individual substitutions affect the affinity of the Ab, Ab to the TSHr are of far greater pathological significance than those to Tg and TPO, but the current repertoire of Ab to the TSHr has yet to include the high affinity IgG Ab characteristic of patient serum Ab, Instructive analysis of the role of somatic hypermutation in the development of TSHr Ab therefore still awaits the isolation of the pathologically active repertoire. Despite this, the Ab response in thyroid autoimmunity remains one of the best characterised of human autoimmune diseases.     

Somatic hypermutation and B-cell malignancies

During a follicle centre response, the immunoglobulin genes are subjected to a hypermutation mechanism which introduces predominantly single base changes, non-randomly, into the immunoglobulin V region (IgV) genes. B cells with mutated IgV genes are then selected according to the affinity of the encoded antibody for antigen retained on the follicular dendritic cells, resulting in an increase in the affinity of the humoral response. The identification of mutated immunoglobulin genes has been applied to the study of normal B cells and B-cell lymphomas to determine either follicle centre cell ancestry, or continued influence of the follicle centre microenvironment. Although analysis of mutations in many lymphomas has confirmed previous hypotheses, there have been some surprises, such as the identification of rearranged and mutated IgV genes in Hodgkin's Reed–Sternberg cells. In this mini-review we will examine the characteristics of the hypermutation mechanism and the way in which mutations in IgV genes have been used to study B-cell malignancies. Copyright © 1999 John Wiley & Sons, Ltd.     

Somatic hypermutation: subverted DNA repair

Somatic hypermutation generates high-affinity antibodies of different isotypes that efficiently protect us against a plethora of pathogens. Recent analyses of the types of mutations produced in gene-deficient mice have indicated how DNA repair proteins are drawn into the pathway. Activation-induced cytosine deaminase begins the process by deaminating cytosine to uracil in DNA. The uracils are then recognized by the base excision repair protein uracil DNA glycosylase and by the mismatch repair proteins MutS homologue 2 and MutS homologue 6. Instead of repairing the uracils, these proteins attract low fidelity DNA polymerases, which synthesize nucleotide substitutions at an unprecedented level.     

Somatic hypermutation and mismatch repair in non-B cells

Mismatch repair contributes to hypermutation in B lymphocytes, both by increasing the frequency of mutations and by changing the mutational patterns. In this paper, we investigated whether or not mismatch repair influences activation-induced cytidine deaminase (AID)-mediated hypermutation in a non-B lymphocyte line. We did so by regulating expression of MutL homologue MLH 1, which is essential in mismatch repair, in a kidney cell line that had been transduced by an AID-containing vector. Whether or not MLH1 was expressed, we found no difference in the mutation rates of an indicator gene. We conclude that in order to contribute to hypermutation, mismatch repair needs additional factors that are present in activated B lymphocytes, but absent in the cell line investigated.     

Somatic hypermutation of immunoglobulin genes in human neonates

The antibody response in the young infant is limited in several ways; in particular, responses generally are of low affinity and restricted to IgM. This raises the question whether the affinity maturation process, consisting of somatic mutation of immunoglobulin genes coupled with selection of high-affinity variants, is operative in the neonate. Re-arranged V_H6 genes were amplified by polymerase chain reaction (PCR) from cord blood and from peripheral blood of infants. Heteroduplex analysis detected mutation in only 2/18 cord blood samples, while mutations were seen from about 10 days of age onwards. Cloning and sequencing of mutated neonatal V_H6 genes showed that mutated sequences contained relatively few mutations (one to three mutations per sequence) compared with published values of about 10 in adult IgM sequences. Selection was not evident in the majority of neonatal samples. Thus mutation can occur in the human neonate, but is minimal and generally not accompanied by selection. The age at which affinity maturation develops effectively is yet to be defined.     

Somatic hypermutation in normal and transformed human B cells

Summary: In the human, most IgM⁺IgD⁺ as well as CD5* peripheral blood B cells express unmutated V genes and thus can be assigned to a pre-germinal centre (GC) stage of development. The memory B-cell compartment generated in die GC reaction and characterized by cells bearing somatically mutated V-region genes consists not only of class-switched cells, but also of lgM-only B cells and perhaps a subset of IgM⁺IgD⁺ B cells expressing the CD27 antigen. Comparison of the rearranged V-region genes of human B-cell lymphomas with those of the normal B-cell subsets allows the identification of the progenitor cells of these tumours in terms of their stage of maturation. On this basis, most B-cell on-Hodgkin lymphomas, and in addition Hodgkin and Reed-Stern berg (HRS) cells in Hodgkin's disease (HD). are derived from B cells ac a GC or post-GC stage of development. The mutation pattern indicates that the precursors of the tumour clones have been stringently selected for expression of a functional antigen receptor with one notable exception: HRS cells in classical (but: not lymphocyte-predominant) HD appear to be derived from "crippled" GC B cells. Sequence analysis of rearranged V genes amplified from single tonsillar GC B cells revealed that the somatic hypermutation process introduces deletions and/or insertions into V-region genes more frequently that indicated by previous investigations. Presumably, this feature of the hypermutation mechanism is often responsible for the generation of heavy chain disease, and also several types of chromosomal translocations of oncogenes into immunoglobulin loci in human B-cell lymphomas.     

A Human Monoclonal IgG1λ Anti-Hepatitis B Surface Antibody

Peripheral blood mononuclear cells from a donor with a high titre of anti-hepatitis B surface (HBs) antibodies were fused with a cell line that was positive for Epstein-Barr virus nuclear antigen and sensitive to hypoxanthine-aminopterine-thymidine. A cell line was established that produces a monoclonal IgG1 lambda anti-HBs antibody. Afterwards, it appeared that the anti-HBs antibody-producing cell line had arisen from Epstein-Barr virus transformation of the donor B cells. The cell line is capable of producing up to 60 micrograms/ml of the monoclonal antibody, which has a high avidity for HBs antigen (Ag) and recognizes both ad and ay subtypes. The antibody is useful as a reagent for the detection of HBsAg in human serum. Over 1000 patient sera have been tested with a conventional third-generation assay in parallel, and only a single discrepant serum was found.     

Antibody Responses to λ1J558 and λ2315 Light Chains

Specificity of BALB/c antibody responses to lambda chains of isologous myeloma proteins 315 and J558 was explored by enzyme-linked immunosorbent assay. lambda-chain binding antibodies were not detected when immunizing with assembled (H + L) myeloma proteins. However, relatively high titred IgG antibodies were elicited by free lambda 2(315) immunization. Antibodies were directed to 'hidden' determinants since binding was abrogated upon H + L assembly of chains. At least a portion of antibodies bound antigenic determinants in the variable region and cross-reacted with lambda 1 land lambda 3 chains. Free lambda 1J558 immunization induced low-titred, predominantly IgM antibodies that also only reacted with 'hidden' determinants. These determinants were most probably located in the constant (C) region and no cross-reaction to lambda 2 or lambda 3 was observed. An artefact of technical importance was noted: myeloma proteins exposed 'hidden' determinants on their lambda chains when coated directly to polystyrene walls. This artefactual exposition was lost when anti C-region antibody spacer molecules were inserted between the wall and the myeloma proteins. Antibody and T helper cell (Th) responses to free lambda 2(315) covaried significantly in various strains while antibody and Th responses to free lambda 1J558 did not. In some strains, weak antibody responses were detected without detectable Th.     

Amyloidosis Related to a λ IV Immunoglobulin Light Chain Protein

Amyloid subunit proteins related to the lambda IV subgroup of immunoglobulin light chains have not been previously reported. We have determined the amino acid sequence of an AL amyloid protein BAK and shown that it has the structure typical of lambda IV light chain proteins. This protein, which was isolated from the spleen of a patient with AL amyloidosis, has 111 residues in the variable domain and also includes the first tryptic peptide of the constant domain for a total of 130 residues. Comparison of the primary structure of this protein with the only other completely characterized lambda IV protein (SH) reveals that they are highly homologous with only one amino acid change in FR1, two changes in FR2, and one change in FR3. The CDR regions also show few changes, with only three in CDR1, one in CDR2, and five in CDR3. To test the hypothesis that the formation of AL amyloid is related to changes in the FR regions which could affect molecular aggregation, the structure of BAK was compared with the myeloma protein SH with respect to the presumed tertiary structure. Only limited amino acid substitution was found in the surface positions that might affect intradimer and interdimer aggregation. These included an isoleucine for leucine change at position 43 and phenylalanine for valine at 45, which may affect intradimer interaction and a change of histidine to asparagine at position 67.     

Somatic hypermutation defects in two adult hyper immunoglobulin M patients

Immunologic Research - Hyper immunoglobulin M (HIGM) syndrome is a rare disorder of the immune system with impaired antibody functions. The clinical picture of the patients varies according to the...     

Fine-Specificity of the λ and χ L Chains Associated with Antibodies Directed to α(l→3) Glucosyls in Dextran

The function of BALB/c primary B precursors, responding to dextran (dex) B-1355, and the fine-specificity of the B-1355-binding, lambda and chi, monofocal antibody (Ab) generated by the precursors have been examined. In splenic fragments from Limulus polyphemus haemocyanin (LPH)-primed, lethally irradiated, euthymic or nu/nu BALB/c mice cultured with thymus-independent (TI) dex B-1355, B-1355-lipopolysaccharide (LPS), B-1355-LPS-LPH, or thymus-dependent (TD) dex B-1355-LPH, the lambda 1 precursors responded with B-1355-binding Ab substantially equally with respect to precursor frequency, rate of Ab production, and range of fine-specificity, but not with respect to frequency of the IdX and IdI isotypes related to the VH and DH associated with the lambda 1. The lambda 2 contributed minimally to the repertoire. The chi precursors responded with B-1355-binding Ab at a rate nearly equal to the lambda 1 only under TD stimulus in euthymic fragments. A comparison of the lambda 1 and chi Ab fine-specificity, by inhibition of binding with dex differing in epitope contents and configurations, showed marked restriction in the chi relative to the lambda 1. Only approximately 10% of the relatively more abundant chi TD response showed fine alpha (1 leads to 3) specificity similar to that of the lambda 1. The lambda 1 fine-specificity diversity resided mainly in the IdI fraction.     

N-Terminal Amino Acid Sequence of Amyloid Fibril Protein AR, Prototype of a New λ-Variable Subgroup, VλV

An amyloid fibril protein AR has been further characterized as to amino acid sequence of the first 45 N-terminal residues. There are clear homologies with light chain-variable regions, particularly with λ light chains and mostly with the VλII subgroup. However, there is as much as a 55% difference from the Vλ.II subgroup and even greater differences from other subgroups. This is much more than the variation known to occur within a given light chain-variable subgroup and also a greater difference than those known to occur between the existing subgroups. For this reason protein AR appears to be the prototype for a new variable subgroup of λ chains. In agreement with the National Biomedical Research Foundation, this new subgroup is termed VλV.     

Monoclonal Antibodies Specific for Variable and Constant Domains of Murine λ Chains

Rat monoclonal antibodies directed against the BALB/c myeloma protein M315 (α,λ2) are described. 9A8 (IgG1) binds the V domain of λ2 and cross-reacts with λ1 and λ3 chains. 2B6 (IgG2a) is directed to the C domain of λ2 and cross-reacts λ3. The antibodies bind isolated chains as well us complete immunoglobulins. The monoclonals detect soluble immunoglobulin (radioimmunoassay), immunoglobulin immobilized on polystyrene (enzyme-linked immunosorbent assay), immunoglobulin bound to nitrocellulose (immunoblotting), and surface immunoglobulin intercalated in cell membranes (immunofluorescence). The antibodies are easily purified on protein G immunosorbents and may be biotinylated or conjugated with fluorescein isothiocyanate without loss of capacity to bind. In addition to the anti-λ antibodies, a C_α2/C_α3-specific monoclonal antibody, 8D2 (IgG2a) is described.     

Protein inheritance: λ plasmid replication perpetuated by the heritable replication complex

Background : Replication of a plasmid derived from the Escherichia coli phage λ initiates by binding of the λO protein initiator to the origin of λ DNA replication, ori λ. The λP protein participates in subsequent steps of assembly of the λ replication complex. A function of λP required for replication complex assembly is inactivated at 43°C by the ts1 mutation.
Results : We found that the λ replication complex assembled at 30°C survives the temperature upshift in λ cro ts P ts1 plasmid-harbouring bacteria. We present several lines of evidence that in this system (in which the replication complex assembly does not occur), the replication complex assembled prior to the temperature upshift is inherited by one of two daughter plasmid copies at each replication round for more than 30 cell generations. The 'old' replication complex-driven replication is chloramphenicol-resistant and rifampicin-sensitive. This replication is dependent on λO and host dnaK , dnaJ and grpE chaperone gene functions.
Conclusions : The λO-containing replication complex is inherited together with DNA and bears information how to initiate the next round of replication at ori λ; thus, we consider that this phenomenon deserves to be called protein inheritance.     

Distribution of κ and λ Light Chain Isotypes among Human Blood Immunoglobulin-Secreting Cells after Vaccination with Pneumococcal Polysaccharides

The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pokeweed mitogen (PWM) and Epstein-Barr virus (EBV), IgM-, IgG- and IgA-secreting cells expressed the kappa light chain isotype in approximately 65% of the cells. IgM- and IgG-secreting cells induced by vaccination with pneumococcal polysaccharides had a similar percentage of kappa light chain-containing cells. In contrast, IgA-secreting cells induced by vaccination with pneumococcal polysaccharides showed a different (bimodal) distribution as regards expression of kappa light chain. The majority (56%) of the investigated individuals expressed kappa light chain in approximately 50% of the cells and the rest expressed kappa light chains in approximately 80% of the cells. The percentage of cells containing kappa light chains among spontaneous IgA-secreting cells in unvaccinated individuals was approximately 50% and thus also differed from the general pattern for mitogen-activated B cells. The light chain pattern of IgA-secreting cells from individuals vaccinated with pneumococcal polysaccharides and from unvaccinated individuals probably indicates that these cells are being derived from B-cell clones with a limited idiotypic heterogeneity, which have been selected and clonally expanded by naturally occurring antigens at the mucosal membranes.