The analysis of organization and diversity mechanism in goat immunoglobulin light chain gene loci

The genomic organization of goat immunoglobulin light chains (Igλ and Igκ) loci were annotated based on the goat genome database. The goat Igλ chain located on chromosome 17 contains at least 35 V_λ gene fragments (seven potential functional genes, one ORF and 27 pseudogenes), two J_λ-C_λ clusters arranged in a V_λ(35)-J_λ2-C_λ1-J_λ1-C_λ2 pattern, with another C_λ3 on scaffold. The Igκ locus included 11 Vκ (five potential functional genes, two ORFs and four pseudogene fragments), three J_κ genes and a single C_κ gene ordered in V_κ(35)-J_κ(3)-C_κ pattern on chromosome 11. By analyzing the clonies of Igλ and Igκ, we further found V_λ2 (26.23 %) & V_λ3 (73.11 %), V_κ2 (52.07 %) & V_κ4 (46.15 %) were predominately used in the expression of λ and κ chains respectively. λ chain showed more abundance in connective diversity than κ chain. Besides, somatic hypermutation with higher frequency in both immunoglobulin light chains was the major mechanism for the goat repertoire diversity. These results demonstrated goat immunoglobulin light chain variable region genome loci and repertoire diversity.     

Multiple germline functional VL genes contribute to the IgL repertoire in ducks

In the immunoglobulin light chain gene loci of nearly all bird species examined to date, there is only a single functional variable gene segment that can recombine with joining gene segments. Thus, Ig light chain diversity relies on gene conversion using pseudogenes as sequence donors to modify the single rearranged variable gene. In the present study, we have sequenced a bacterial artificial chromosome (BAC) clone containing the entire duck Igλ light chain gene locus. Although only a single pair of J_λ and C_λ was found, 88 V_λ gene segments were identified upstream of the J_λ and C_λ segments. Among the identified V_λ gene segments, 79 appear to be pseudogenes, the remaining 9 are structurally intact and all are able to functionally rearrange with the J_λ. Phylogenetic analyses suggest that the 9 functional variable genes may have been derived from a single gene through duplication events. Although these multiple functional variable gene segments can be subject to VJ recombination, both gene conversion and somatic hypermutation are also actively involved in the generation of diversity in duck Igλ light chains. These data provide significant insight into understanding the duck Ig system.     

Somatic hypermutation of VHS107 genes is not associated with gene conversion among family members

Murine B cells proliferating in the germinal centers of peripherallymphoid tissue accumulate mutations in their rearranged variableregions, a diversification process which contributes to affinitymaturation of the antibody response. The highly targeted natureof the hypermutation process could be explained by a somaticgene conversion mechanism. Well characterized examples of suchan activity in B cells are seen during diversification of thechicken and rabbit Ig repertoires. The genomic organization,low complexity and high degree of homology exhibited by thefour members of the murine V_HS107 gene family suggested thatthese gene segments may be suitable candidates for the searchof gene conversion events derived from upstream V_HS107 counterparts.After an immune response to a complex T cell-dependent antigen(sheep red blood cells), rearranged V13, V11 and V1 genes wereisolated from splenic extrafollicular and germinal center Bcells. Extensive somatic mutation was evident in V11 and V1sequences. When these sequences were examined, as well as V1sequences isolated from phosphorylcholine-specific hybridomas,the observed nucieotide changes were not associated with anygene conversion between family members, suggesting instead thatthey arose by a mechanism which introduces point mutations.     

Light chain variable (VL) sequences of rheumatoid factors (RF) in patients with primary Sjögren's syndrome (pSS): moderate contribution of somatic hypermutation

We have characterized and sequenced the variable (V) region genes of the light (L) chains of 10 immunoglobulin (IgM) rheumatoid factor (RF) monoclonal antibodies (MoAb) derived by the hybridoma/Epstein-Barr virus (EBV) technique from the peripheral blood of patients with primary Sjögren's syndrome (pSS). Six out of 10 RFs used lambda (λ) L chains, while four RFs used kappa (κ) L chains. Five out of the six λ RFs were encoded by V_λ3 gene segments, the sixth one was encoded by a V_λ1 gene segment. This preferential utilization of the V_λ3 family genes suggests selective expansion of the B cell in pSS. Three of the κ RFs used V_κ3 gene segments, while the fourth used a V_κ2 gene segment. Half of the RFs were found as unmutated copies of their closest germline (GL) gene. Interestingly these RFs were previously shown to use heavy (H) chains in GL gene configuration. Three RFs have very few mutations (2–3) and only two RFs have substantial numbers of mutations (6 and 11). This also correlated with the number of mutations in the respective H chains. In contrast to RFs in normal and RA these results further suggest the somatic mutation to be of moderate importance in the generation of RF from the peripheral blood of pSS patients.     

Subtle differences in antibody responses and hypermutation of λ light chains in mice with a disrupted χ constant region

Analysis of λ light chain use in normal mice is made difficult by the dominant x light chain repertoire. We produced mice rendered deficient in x light chain expression by gene targeting and focused on questions concerned with the generation of λ light chain diversity. Whilst these mice compensate the x deficiency with increased λ titers, and their Ig level is therefore not significantly reduced, they show major differences in immunization titers, germinal center (GC) development and somatic hypermutation. After immunization, using antigens that elicit a restricted IgL response in normal mice, we obtained in the x^?/? mice elevated primary antibody titers but a subsequent lack in titer increase after repeated antigen challenge. Analysis of the Peyer's patches (PP) revealed a dramatically reduced cell content with rather small but highly active GC. Flow cytometric analysis showed different cell populations in the PP with enriched peanut agglutinin (PNA)^hi/CD45R(B220)⁺ B cells, implying that the apparent compensation for the lack of x light chain expression involves the GC microenvironment in cell selection, the initiation of hypermutation and high affinity expansion. The three Vλ genes, V1, V2 and Vx, are mutated in the GC B cells, but show no junctional diversity. In contrast, a reduced rate of Vλ hypermutation is found in the hybridoma antibodies, which appears to reflect a selection bias rather than structural constraints. However, mechanisms of somatic mutation and specificity selection can operate with equal efficiency on the few Vλ genes.     

The VH repertoire and clonal diversification of B cells in inflammatory myopathies

The contribution of antigen‐driven B‐cell adaptive immune responses within the inflamed muscle of inflammatory myopathies (IMs) is largely unknown. In this study, we investigated the immunoglobulin V_H gene repertoire, somatic hypermutation, clonal diversification, and selection of infiltrating B cells in muscle biopsies from IM patients (dermatomyositis and polymyositis), to determine whether B cells and/or plasma cells contribute to the associated pathologies of these diseases. The data reveal that Ig V_H gene repertoires of muscle‐infiltrating B cells deviate from the normal V_H gene repertoire in individual patients, and differ between different types of IMs. Analysis of somatic mutations revealed clonal diversification of muscle‐infiltrating B cells and evidence for a chronic B‐cell response within the inflamed muscle. We conclude that muscle‐infiltrating B cells undergo selection, somatic hypermutation and clonal diversification in situ during antigen‐driven immune responses in patients with IMs, providing insight into the contribution of B cells to the pathological mechanisms of these disorders.     

Human IgA- and IgM-secreting intestinal plasma cells carry heavily mutated VH region genes

Single IgA- or IgM-secreting plasma cells were isolated from histological sections of human jejunum and terminal ileum, and Ig heavy chain variable (V_H ) region genes were amplified and sequenced. Taken together, 62 of 63 cells analyzed harbored somatically mutated V_H region genes, indicating that the vast majority of both IgA- and IgM-secreting intestinal plasma cells derive from germinal center B cells. On average, rearranged V_H genes of IgA- and IgM-secreting plasma cells showed a mutation frequency of 9.0 % and 8.5 %, respectively, which exceeds the level of somatic mutation of V region genes carried by human memory B cells. Moreover, we detected deletions or insertions in the complementarity-determining regions of 5 of the 58 functional V_H region genes analyzed, suggesting that these alterations may contribute to the diversification of the human antibody repertoire in the course of an immune reaction.     

Immunization with immune complex alters the repertoire of antigen-reactive B cells in the germinal centers

The differentiation of memory B cells in germinal centers (GC) is selectively enhanced upon administration of antigen-antibody complexes. To characterize the repertoire of this response, we examined the rearranged immunoglobulin heavy chain variable (V_H) genes from mouse splenic GC after a single immunization with either antigen, nitrophenyl (NP) hapten coupled to keyhole limpet hemocyanin, or with a preformed complex of antigen with a monoclonal anti-NP antibody of γ1 isotype. Among antigen-immunized mice, NP-reactive GC B cell populations in the antigen-induced GC consisted mostly of cells expressing the canonical V186.2 gene which contained, on average, 0.8 point mutations/V_H gene by day 8 after immunization. These results are indicative of the beginning of somatic hypermutation and consistent with previously published analyses of NP antigen-driven GC. In contrast, the NP-specific B cells in GC that were elicited by administration of immune complex represented a heterogeneous cell population expressing nine different germ-line segments of the V186.2/V3 (J558) gene family, i.e. V23, V24.8, C1H4, V3, CH10, V165.1, V102, V671.5 and V186.2. Moreover, the average frequency of mutations in these genes was 1.7, reaching up to 4 mutations/V_H in some GC. Administration of the antigen NP in complex with specific antibody apparently alters the process of interclonal competition in the GC and results in loss of dominance by V186.2⁺ cells and nearly stochastic representation of diverse clono-types. These results suggest an important feedback regulation of the B cell repertoire by antibody and indicate a role for immune complexes in the activation of somatic hypermutation.     

Molecular Ig gene analysis reveals that monocytoid B cell lymphoma is a malignancy of mature B cells carrying somatically mutated V region genes and suggests that rearrangement of the kappa-deleting element (resulting in deletion of the Ig kappa enhancers) abolishes somatic hypermutation in the human

Five cases of monocytoid B cell lymphoma (MBCL) were analyzed for somatic mutations in the rearranged V region genes. Somatic mutations were found in four of the five cases, whereas one unusual CD5⁺ lymphoma harbored unmutated V region genes. Since somatic mutations are introduced into V regiongenes of antigen-activated B cells in the course of T cell-dependent immune responses, these results suggest a derivation of the tumor B cells in MBCL from antigen-experienced mature B cells. An analysis of the ϰ-deleting element in two of the cases in which mutated V_H but unmutated and nonfunctional V_ϰ gene rearrangements were found suggests that somatic hypermutation does not take place in human rearranged V_ϰ region genes when the C_ϰ gene and the ϰ enhancers have been deleted in cis by rearrangement of the ϰ-deleting element.     

Primary central nervous system lymphomas are derived from germinal-center B cells and show a preferential usage of the V4-34 gene segment

Primary central nervous system lymphomas (PCNSLs) have recently received considerable clinical attention due to their increasing incidence. To clarify the histogenetic origin of these intriguing neoplasms, PCNSLs from 10 HIV-negative patients were analyzed for immunoglobulin (Ig) gene rearrangements. All tumors exhibited clonal IgH gene rearrangements. Of the 10 cases, 5 used the V4-34 gene segment, and all of these lymphomas shared an amino acid exchange from glycine to aspartate due to a mutation in the first codon of the complementarity-determining region 1. No preferential usage of D(H), J(H), V(kappa), J(kappa), V(lambda), or J(lambda) gene segments was observed. All potentially functional rearrangements exhibited somatic mutations. The pattern of somatic mutations indicated selection of the tumor cells (or their precursors) for expression of a functional antibody. Mean mutation frequencies of 13. 2% and 8.3% were detected for the heavy and light chains, respectively, thereby exceeding other lymphoma entities. Cloning experiments of three tumors showed ongoing mutation in at least one case. These data suggest that PCNSLs are derived from highly mutated germinal-center B cells. The frequent usage of the V4-34 gene and the presence of a shared replacement mutation may indicate that the tumor precursors recognized a shared (super) antigen.     

Synovial IgG rheumatoid factors show evidence of an antigen-driven immune response and a shift in the V gene repertoire compared to IgM rheumatoid factors

We have established IgG rheumatoid factor (RF)-secreting hybridoma cell lines from the synovial tissues of three patients from whom we have previously characterized several IgM RF. The IgG RF bind human and rabbit IgG and form intracellular complement-fixing complexes indicative of a self association process in vivo. Nucleotide sequence analysis revealed that two IgG RF used V_HIII gene segments, while one used a V_HI gene segment. The V_L gene usage consisted of a Vχ1, a Vλ2 and a Vχ4/Vχ6 hybrid, confirming our previous findings that many different V_L genes can contribute to RF specificity. Although the IgG RF used V_H genes from the same families that dominated the IgM RF response, two of the actual gene segments employed were not found among the IgM RF. In contrast to IgM RF, which in general were not very mutated, the IgG RF showed somatic mutations characteristic of an antigen-driven immune response.     

Immunoglobulin VH Gene Mutational Analysis Suggests that Primary Effusion Lymphomas Derive from Different Stages of B Cell Maturation

Primary effusion lymphoma (PEL) is a recently described distinct subtype of non-Hodgkin’s lymphoma associated with infection by the Kaposi’s sarcoma-associated herpesvirus, also called human herpesvirus-8. Most cases of PEL are also associated with the Epstein-Barr virus (EBV). In order to better characterize the cellular origin of PEL, we investigated the immunoglobulin (Ig) heavy chain variable region (V_H) genes expressed by tumor cells of the BC-1 and BC-3 cell lines derived from PELs and five original PEL specimens. In the six EBV-positive PELs examined, including the BC-1 cell line, the expressed V_H gene sequences showed numerous point mutations relative to the putative germline V_H gene sequences. In addition, the V_H segment of one of these cases showed intraclonal sequence heterogeneity, indicating ongoing somatic mutation. In five cases, the distribution and type of mutations indicated that tumor cells had been selected by antigen. Because somatically mutated Ig genes are expressed by B cells that have reached a germinal center/post-germinal center stage of development, these findings suggest that the PEL cell of origin is a germinal center or post-germinal center B cell in most cases. In contrast, the V_H gene segment expressed by tumor cells of the BC-3 cell line, which was originated from an EBV-negative PEL obtained from an HIV-negative patient, was unmutated, suggesting a pre-germinal center B cell origin for tumor cells of this particular PEL cell line. Taken together, these findings suggest that development of PELs may not be restricted to one stage of B cell differentiation and may represent transformation of B cells at different stages of ontogeny.     

V(D)J hypermutation and receptor revision: coloring outside the lines

At least three mechanisms increase potential genetic diversity in peripheral B lymphocytes: hypermutation, gene conversion and secondary V(D)J rearrangements. These diversifying activities were once believed to be strictly confined to the immunoglobulin loci and B cells. Recent experiments demonstrate that this is not the case. Hypermutation has now been shown to diversify the BCL-6 genes of germinal-center B cells. The role, if any, of these mutations in the immune response remains unknown but the notion that the hypermutation mechanism is targeted solely to immunoglobulin genes is no longer tenable. Peripheral T cells may also diversify their antigen receptors by the reactivation of RAG (recombination-activating gene)1 and RAG2 and secondary V(D)J rearrangements. These new findings suggest a remarkable genetic plasticity in subsets of antigen-reactive lymphocytes and may frame new questions of clonal selection and self tolerance.     

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V_HDJ_H, VκJκ and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V_HDJ_H, VκJκ, and c-myc gene sequences. Tumor-site specific V_HDJ_H, VκJκ, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V_HDJ_H, VκJκ, and c-myc mutations displayed predominance of transitions over transversions. The “neoplastic” V_HDJ_H sequence was expressed by about 10⁻⁵ cells in the bone marrow, and contained two of the three orbital, but none of the testicular V_HDJ_H mutations. The nature and distribution of the Ig V(D)J mutations found in the κ chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V_HDJ_H, VκJκ, and c-myc genes with comparable efficiency and modalities.     

Available λ B cell repertoire in the mouse: Evidence of positive selection by environmental factors

We have recently shown that, from two BALB/c mice treated with rabbit anti-Cλ2/Cλ3 antibodies coupled to lipopolysaccharide, variable heavy chain (V_H) family repertoires associated with λ2 or λ3 light chains can differ from one λ. subtype to another and from one individual mouse to another. Indeed, 4 out of 6 λ2 (VxJ2) hybridomas from one mouse preferentially expressed the V_H10 family while 3 out of 8 λ2 (V2J2) and 5 out of 8 λ2 (VxJ2) hybridomas from a second mouse preferentially expressed the S107 and VGAM3.8 V_H families, respectively. In this report, we describe the structural basis of such preferential pairings by sequence analysis of the 12 λ2 hybridomas. The sequence comparison of their V_H regions show that each preferential association of a VH family to one Vλ region is restricted to the use of a single member or very closely related members inside a V_H family and that a great variability of CDR3 of heavy chain is observed. We, therefore, suggest that environmental factors can modify the available XλB cell repertoire through a positive selection of particular VH/Vλ pairings. Moreover, our data support that this selection does not require clonal expansion and punctual somatic mutation.     

Molecular analysis of rheumatoid factor (RF)-negative B cell hybridomas from rheumatoid synovial tissue: evidence for an antigen-induced stimulation with selection of high mutated IgVH and low mutated IgVL/λ genes

The mutational pattern of IgV_H and IgV_L genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgV_H genes of the B cell hybridomas belonged to the V_H3 family (DP42; DP47, n = 2; DP53), the V_H1 family (DP75), the V_H4 family (DP71) and the V_H5 family (DP73); 7/7 IgV_H genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgV_H genes and the mean R/S ratio of all IgV_H genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgV_L/λ genes belonged to the Vλ1 family (DPL2, DPL5, DPL8nf), the Vλ2 family (DPL11, n = 2) and to the Vλ6 family (IGLV6S1); 6/6 IgV_L genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgV_L genes and the mean R/S ratio of all IgV_L was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4⁺ follicular dendritic cells and Ki-67⁺ proliferating cells and a dominance of IgA⁺ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgV_H and IgV_L/λ genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.