Immunoglobulin genomics in the guinea pig (Cavia porcellus)

In science, the guinea pig is known as one of the gold standards for modeling human disease. It is especially important as a molecular and cellular biology model for studying the human immune system, as its immunological genes are more similar to human genes than are those of mice. The utility of the guinea pig as a model organism can be further enhanced by further characterization of the genes encoding components of the immune system. Here, we report the genomic organization of the guinea pig immunoglobulin (Ig) heavy and light chain genes. The guinea pig IgH locus is located in genomic scaffolds 54 and 75, and spans approximately 6,480 kb. 507 V(H) segments (94 potentially functional genes and 413 pseudogenes), 41 D(H) segments, six J(H) segments, four constant region genes (μ, γ, ε, and α), and one reverse δ remnant fragment were identified within the two scaffolds. Many V(H) pseudogenes were found within the guinea pig, and likely constituted a potential donor pool for gene conversion during evolution. The Igκ locus mapped to a 4,029 kb region of scaffold 37 and 24 is composed of 349 V(κ) (111 potentially functional genes and 238 pseudogenes), three J(κ) and one C(κ) genes. The Igλ locus spans 1,642 kb in scaffold 4 and consists of 142 V(λ) (58 potentially functional genes and 84 pseudogenes) and 11 J(λ) -C(λ) clusters. Phylogenetic analysis suggested the guinea pig's large germline V(H) gene segments appear to form limited gene families. Therefore, this species may generate antibody diversity via a gene conversion-like mechanism associated with its pseudogene reserves.     

A multifunctional element in the mouse Igκ locus that specifies repertoire and Ig loci subnuclear location

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes.     

Similarities and differences between the light and heavy chain Ig variable region gene repertoires in chronic lymphocytic leukemia

Analyses of Ig V(H)DJ(H) rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V(L) and J(L) gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V(L) genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V(L) and J(L) segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V(H) repertoire, V(L) gene use was not significantly different than that of normal B-lymphocytes. In addition, Vkappa genes that lie more upstream on the germline locus were less frequently mutated than those at the 3' end of the locus; this was not the case for Vlambda genes and is not for V(H) genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.     

Stepwise activation of the immunoglobulin mu heavy chain gene locus.

The immunoglobulin heavy chain (IgH) gene locus spans several megabases. We show that IgH activation during B-cell differentiation, as measured by histone acetylation, occurs in discrete, independently regulated domains. Initially, a 120 kb domain of germline DNA is hyperacetylated, that extends from D(FL16.1), the 5'-most D(H) gene segment, to the intergenic region between Cmu and Cdelta. Germline V(H) genes were not hyperacetylated at this stage, which accounts for D(H) to J(H) recombination occurring first during B-cell development. Subsequent activation of the V(H) locus happens in at least three differentially regulated domains: an interleukin-7-regulated domain consisting of the 5' J558 family, an intermediate domain and the 3' V(H) genes, which are hyperacetylated in response to DJ(H) recombination. These observations lead to mechanisms for two well-documented phenomena in B-cell ontogeny: the sequential rearrangement of D(H) followed by V(H) gene segments, and the preferential recombination of D(H)-proximal V(H) genes in pro-B cells. We suggest that stepwise activation may be a general mechanism by which large segments of the genome are prepared for expression.     

YY1 controls Igκ repertoire and B‐cell development,and localizes with condensin on the Igκ locus

Conditional knock‐out (KO) of Polycomb Group (PcG) protein YY1 results in pro‐B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino‐acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild‐type YY1 rescued B‐cell development, YY1ΔREPO failed to rescue the B‐cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co‐localize at numerous sites across the Ig kappa locus. Knock‐down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1‐condensin complexes in Igκ locus structure and rearrangement.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related V_L chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V_L chain genes caused by selection and clonal expansion of B cells expressing particular V_L genes. In addition, the data document an accumulation of B cells bearing mutated V_L gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Surface immunoglobulins on mouse myeloma cells.

Plasma cells from 22 different transplantable mouse myelomas (PCT) were tested by 14 different class-specific and type-specific anti-immunoglobulin antiserums for cytotoxicity effects using a trypan blue-exclusion method. Seven IgF,κ tumors were all sensitive to anti-IgF and anti-κ antiserums. None of the other antiserums showed a cytotoxic effect. Five IgG,κ and four IgH,κ tumors were lysed by anti-κ serums, but not by anti-IgG or anti-IgH or the others. One of two IgA,κ tumors was lysed by anti-κ, but neither was lysed by anti-IgA or the other serums.One IgM,λ tumor was lysed by anti-IgM and anti-λ. Two λ Bence Jones tumors were not lysed by anti-λ, but both of these and the IgM,λ tumor were lysed by anti-κ. One κ Bence Jones tumor was lysed only by anti-κ serum.Only the IgF tumors and an IgM tumor were lysed by the appropriate anti-heavy chain serum, and $\text{21}\text{22}$ lines had κ surface determinants.     

The evolution of multiple isotypic IgM heavy chain genes in the shark

The IgM H chain gene organization of cartilaginous fishes consists of 15-200 miniloci, each with a few gene segments (V(H)-D1-D2-J(H)) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer loci, and characterized the IgH isotypes for organization, functionality, and the somatic diversification mechanisms that act upon them. Gene numbers differ slightly between individuals ( approximately 15), but five active IgM subclasses are always present. Each gene undergoes rearrangement that is strictly confined within the minilocus; in B cells there is no interaction between adjacent loci located > or =120 kb apart. Without combinatorial events, the shark IgM H chain repertoire is based on junctional diversity and, subsequently, somatic hypermutation. We suggest that the significant contribution by junctional diversification reflects the selected novelty introduced by RAG in the early vertebrate ancestor, whereas combinatorial diversity coevolved with the complex translocon organization. Moreover, unlike other cartilaginous fishes, there are no germline-joined VDJ at any nurse shark mu locus, and we suggest that such genes, when functional, are species-specific and may have specialized roles. With an entire complement of IgM genes available for the first time, phylogenetic analyses were performed to examine how the multiple Ig loci evolved. We found that all domains changed at comparable rates, but V(H) appears to be under strong positive selection for increased amino acid sequence diversity, and surprisingly, so does Cmicro2.