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.     

Analysis of individual immunoglobulin λ light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse λ light chain genes which arose in response to a specific antigen. Laboratory mice possess three Vλ subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single λ light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V_λ1 genes from these single cells for mutational analysis. None of the 32 V_λ1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other Vλ subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.     

Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.     

Cloning and sequencing of human immunoglobulin λ. gene segments

To provide the building blocks for making synthetic antibody fragments we have used the polymerase chain reaction (PCR) to clone human variable (V) gene segments of λ light chains. The PCR primers were based on the sequences of known human Vλ segments, and were used to isolate 14 new Vλ segments (including 4 pseudogenes) from a single individual. We have compiled a sequence directory from this data and other sources to include all known human Vλ segments with open reading frames and we have identified a new Vx family (Vλ IX). Almost all of the segments (22/24) have different sequences in the complementarity-determining regions, setting a lower limit to the structural diversity of the antigen binding sites encoded by human Vλ genes in the human population.     

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or ? and L2 or σ, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross-hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen-stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80 % identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity-determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ-line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J- C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ-line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V-J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.     

Evolutionary comparison of the avian IgL locus: combinatorial diversity plays a role in the generation of the antibody repertoire in some avian species

Immunoglobulin light chain (IgL) diversity is generated in the chicken by recombination between the single functional variable (VL) and joining (JL) gene segments and subsequent somatic diversification of the rearranged VL region. In order to determine whether these events are a general feature of avian IgL genes, we analyzed the organization and recombinatorial characteristics of the IgL loci of several other avian species. Southern blot analysis of bursal and germline DNA using chicken VL and constant (CL) probes revealed that the IgL loci of quail, mallard duck, pigeon, turkey, cormorant, and hawk consist of a family of VL elements, but undergo a single major rearrangement event similar to that observed in chickens. In contrast, several rearrangements were observed in the Muscovy duck locus. A phage clone containing a 26 kb insert that hybridized to VL and CL probes was isolated from a Muscovy duck erythrocyte DNA genomic library. Nucleotide sequencing revealed that the clone contained a single JL-CL region flanked on the 5' side by five VL segments. Unlike the chicken, two of the VL segments (VL1, VL5) appear to be functional. The remaining three VL segments are pseudogenes that lack promoter and leader sequences, but one of these (psi VL3) has recombination signal sequences. Overall, these data indicate that rearrangement of one VL gene segment is a general feature of the IgL locus in many avian species. In these species, the presence of a family of VL elements that do not rearrange suggests that a pseudogene pool may be available for somatic diversification by gene conversion. The organization of the Muscovy duck IgL locus suggests that additional combinatiorial diversity has evolved independently in some avian species.     

Influence of the nude and X-Iinked immune deficiency genes on expression of × and λ light chains

The relative amounts of Igx and Igλ₁ anti-2,4-dinitrophenyl antibodies were measured at various times after immunizing mice with prototype thymus-dependent (TD), thymus-independent type 1 (TI-1) and thymus-independent type 2 (TI-2) antigens. Similar amounts of Igλ₁ were produced after TD and TI-2 immunization and somewhat less was produced after a TI-1 stimulus. In contrast, Igx levels were much greater after TD than after TI-1 or TI-2 antigen. The amount of light chain isotype produced appeared to depend on the molecular form in which the hapten was presented, although possible adjuvant effects were not ruled out. Levels of Igx and Igλ present in nonimmune sera were measured in normal, xid and nude mice. The x/λ, ratio was higher in xid than in normal mice and the difference was demonstrated by F₁ analysis to be due to an X-linked gene. Conversely, the x/λ ratio was lower in nude than in normal mice. This was true for the CBA/Tufts (Igh^j), CBA. Igh^b and C57BL/10 strains. However, there were no detectable differences in the relative frequencies of surface Igx- and Igλ -bearing B cells in adult CBA/Tufts, CBA/N and nude mice. Hence, serum ratios may reflect differences at the level of B cell triggering. Two possible explanations for these differences are discussed. Igx and Igλ may be expressed on functionally distinct B cell subsets. (For instance Igλ -producing cells might be readily triggered by T1 antigens whereas Igx-producing cells are more dependent on T cell signals. Such functional subsets could be determined by light chain expression). Alternatively, cells producing Igx antibody are selected for because they have a higher affinity for antigen. If so, triggering of cells producing high affinity Igx or their subsequent selection is T cell-dependent.     

The Primary Antibody Repertoire of κ-Deficient Mice is Characterized by Non-Stochastic Vλ1 + VH Gene Family Pairings and a Higher Degree of Self-Reactivity

We have investigated the primary antibody repertoire of genetically manipulated 129/Sv κ-deficient (JC_κD) mice, in order to understand the contributions of the λ-light chain, in the absence of an otherwise predominant κ-light chain, to the development of humoral immunity. The expression of V_λ1 gene (λ₁ and λ₃ subtypes) and the V_λ1 + V_H (J558, 36–60, V_H11 and S107) gene family associations were studied in 7.43 × 10³ mitogen-activated splenic B-lymphocyte clones of JC_κD origin. Furthermore, the functional significance of the exclusive expression of the λ-light chain, in the peripheral B-cell repertoire of JC_κD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B-lymphocyte clones in the peripheral B-lymphocyte repertoire. These experiments revealed that: first, of the three available V_λ genes at the λ locus, the V_λ1 gene is the one that is expressed most frequently (59.9%); second, non-random V_λ1 + V_H (J558, 36–60) gene family pairings occur in κ-deficient mice; and third, a higher degree of self-reactivity is generated as a result of exclusive use of the λ-light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B-lymphocyte clones in κ-deficient (129/Sv JC_κD) mice. These observations suggest that the high murine κ/λ ratio in mice may, apart from high sequence diversity at the κ-locus, be a result of endogenous selection against the λ-light chain to restrict self-reactivity within the homeostatic threshold.     

Preparation and characterization of antibodies to the lambda chain variable region (Vlambda) of mouse immunoglobulins.

The variable portion (V_L) of the mouse myeloma protein MOPC-315 (α, λ₂) was obtained from its Fv fragment and was used to immunize rabbits. Anti-V_L antibodies were found to be specific to the V_L region of protein 315 and to precipitate V_L, Fv or light chain but not Fab' or intact M315. Anti-V_L 315 precipitates also the light chain of MOPC-104E (μ, λ₁) but not the intact protein. Intact immunoglobulins (Ig) containing λ chains, although they do not precipitate with anti-V_L, inhibit the binding of anti-V_L to V_L in the radioimmunoassay. Radioimmunoassay inhibition studies demonstrated that V_λ1 and V_λ2 cross-react extensively and that anti-V_L 315 can be used as a general anti-V_λ reagent to detect molecules containing λ chains in mouse serum. Analysis of sera from several mouse strains indicated that V_λ-containing molecules are present in approximately 3% of the Ig population, whereas the SJL strain has no detectable V_λ-bearing molecules. In some of the antisera, anti-V_L 315 slightly cross-reacted with V_K-bearing molecules. Anti-V_L 315 antibodies are not anti-idiotypes since they react with myeloma proteins M315 (anti-2,4-dinitrophenyl MOPC-104E (anti-dextran) and HOPC-1 (specificity unknown) and with most serum Ig containing λ chains. In view of the similarity of mouse λ chain sequences, it is suggested that most anti-V_L 315 is “anti-framework” antibody.     

Immunoglobulin heavy chain variable region analysis in dairy goats

Based on the goat genome database, we have annotated the genomic organization of the goat immunoglobulin heavy chain variable region. The goat IgH locus is present on seven genome scaffolds, and contains ten V_H, three D_H and six J_H segments. After the exclusion of three shorter segments, the V_H genes were divided into two gene families based on sequence similarity. By analyzing the IgH cDNA sequences, we further identified that V_H2 (54.2%), D_H1 (61.7%) and J_H1 (60.5%) segments were most frequently utilized in the expression of the immunoglobulin variable region, and that point mutations introduced by somatic hypermutation were the major mutation present in these expressed variable region. Compared with human and horses, D_H-D_H fusion occurred at a higher frequency in goat V(D)J recombination. These results provided variable insights into goat immunoglobulin heavy chain variable region genome loci and repertoire diversity.     

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.     

Up-regulated recombination-activating gene expression in slg− variants of a human mature B cell line undergoing secondary Igλ, rearrangements in cell culture

To study the role of surface immunoglobulin (sIg) expression in the control of rearrangement activity at the Ig light chain loci, we established rare sIg^? clones (λ^?) from a human sIg⁺ B cell line (μl⁺). Upon expansion of these sIg^? clones, surface immunofluorescence analysis revealed a gradual emergence of sIg⁺ subpopulations, differing from the original tumor cell line both in their idiotypes and Cλ isotypes. DNA analysis revealed that this sIg heterogeneity resulted from a process of ongoing Igλ, rearrangements. That is, one of the Igλ rearrangements in the parental cell line was replaced by novel Igλ rearrangements in the sIg^? clones, which in turn were replaced by yet additional Igλ rearrangements in the sIg⁺ variants. Northern analysis demonstrated that while the expression of the recombination-activating genes RAG1 and RAG2 was relatively low in the parental cell line, their expression was significantly increased in both the sλ g⁺ variants and their sIg⁺ progenies. We thus describe a human mature B cell line, in which differential RAG expression allows sIg heterogeneity to be generated through secondary Igλ gene rearrangements. Our results indicate that the induction of RAG expression may be inversely associated with sIg expression, but that sIg expression, alone, is not sufficient to down-regulate this expression.     

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.     

Immunoglobulin Gene Diversification in Cattle

Research in several species has revealed that different types of mammals have evolved divergent molecular and cellular strategies for generating immunoglobulin diversity. Other chapters in this text have highlighted the specific characteristics unique to chicken, rabbit, mouse, human and sheep B lymphocyte development; namely indicating differences in the mechanisms of diversity and the site of primary B cell development. Studies of the bovine system have indicated that like the sheep system, the ileal Peyer's patch (IPP) is a likely chicken bursal equivalent, and is a site of primary B lymphocyte development. Substantial investigation in sheep has indicated that Ig diversity is created by untemplated somatic mutation and intense selective pressure (Reynaud et al., 1991). The frequency of alteration in the sheep Ig light chain gene locus also is characteristic of the bovine system, however, recent evidence from sequencing of bovine lambda light chain genes indicates that one mechanism that contributes to diversity is gene conversion, utilizing several pseudogenes located in the Ig locus (Parng et al., 1996). The mechanism by which this hyperalteration of Ig genes occurs in both sheep and cattle is poorly understood and is thus the focus of considerable investigation. The study of events in the IPP may also have informative ramifications for secondary diversification of the Ig repertoire by somatic hyperalteration in germinal centers.     

Polymorphism of the functional immunoglobulin variable region genes in the chicken by exchange of sequence with donor pseudogenes

We have isolated a number of new allelic variants of the unique functional genes encoding chicken immunoglobulin heavy and light chain variable regions (V_H1 and V_L1, respectively). The distribution and nature of nucleotide variation among these and previously identified V_H1 and V_L1 alleles demonstrates that random point mutations are likely not the predominant cause of allelic variation at these loci. Comparison of the variant nucleotides with sequences from the pseudo-V_H and pseudo-V_L gene families, which lie 5′ to V_H1 and V_L1, respectively, suggests that the great majority of allelic variants can be accounted for by segmental transfer of sequence from donor pseudogenes into the germ-line VH₁ and V_L1 genes. These results demonstrate that the chicken V_H1 and V_L1 genes are susceptible to sequence replacement at the germ-line level as well as somatically during antibody diversification. The limited repertoire of Bcell specificities produced by gene rearrangement in the chicken has led to speculation that these specificities may play a critical role in the progression of chicken B cell development. The results presented here do not support this hypothesis since many of the allelic variant nucleotides described here encode non-conservative amino acid substitutions within the antigen-binding sites of the Ig molecule.     

The surrogate light chain in B-cell development

The proteins encoded by the V_prB and λ_s genes associate with each other to form a light (L) chain-like structure, the surrogate L chain. It can form Ig-like complexes with three partners - the classical heavy (H) chain, the D_HJ_HC_μ-protein, or the newly discovered p55 chain; these are expressed on the surface of pre-B cells at different stages of development. Here, Friz Melchers and colleagues review the structures of the V_preB and λ_s genes in mouse and their relatives in humans, describe their pattern of expression, and speculate on their possible evolution and functions.     

The human immunoglobulin VH7 gene family consists of a small,polymorphic group of six to eight gene segments dispersed throughout the VH locus

In this report we describe the analysis and mapping of members of the human immunoglobulin V_H7 gene family. V_H7 and V_H1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from V_H1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity-determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162-base pair probe spanning the family-specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The V_H7 gene family was found to be a small but discrete V_H gene family consisting of five to eight germ-line elements, of which at least three are polymorphic. Four different V_H7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence-specific hybridization. Two of the four V_H7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel V_H families. A combination of one and two-dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these V_H7 elements. Individual V_H7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other V_H gene families. The polymorphism of the V_H7 gene segments and their scattered location throughout the V_H locus makes them potentially useful markers for mapping and linkage studies.