The multiple shark Ig H chain genes rearrange and hypermutate autonomously

Sharks and skates are representatives of the earliest vertebrates with an immune system based on V(D)J rearrangement. They possess a unique Ig gene organization consisting of 15 to >50 individual IgM loci, each with one VH, two DH, one JH, and one set of constant region exons. The present study attempts to understand how multiple Ig genes are regulated with respect to rearrangement initiation and to targeting during somatic hypermutation. The linkage of three single-copy IgH genes was determined, and single-cell genomic PCR studies in a neonatal animal were used to examine any relationship between relative gene position and likelihood of rearrangement. Our results show that one to three IgH genes are activated independently of linkage or allelic position and the data best fit with a probability model based on the hypothesis that V(D)J rearrangement occurs as a sequence of trials within the B cell. In the neonatal cell set, two closely related IgH, G2A, and G2B, rearranged at similar frequencies, and their membrane forms were expressed at similar levels, like in other young animals. However, older animals displayed a bias in favor of the G2A isotype, which suggests that although rearrangement at G2A and G2B was randomly initiated during primary repertoire generation, the two very similar IgM sequences appear to be differentially expressed with age and exposure to Ag. We performed genomic single-cell PCR on B cells from an immunized individual to study activation-induced cytidine deaminase targeting and found that hypermutation, like V(D)J rearrangement, occurred independently among the many shark IgH.     

Heavy chain variable region gene families evolved early in phylogeny. Ig complexity in fish

The V regions of channel catfish H chain cDNA clones have been analyzed. Based upon sequence relationships and hybridization analyses, five different groups of VH genes are identified whose definition is consistent with that of five different VH families. Genomic Southern blots indicate that as many as 100 different germ-line VH genes are likely represented by these families. The sequence diversity between identified members of these different families is similar in magnitude to the divergence represented between members of different human or mouse VH families. The FR regions are the most conserved regions when members of different catfish VH families are compared; specific amino acid positions appear to be highly conserved in phylogeny. Equally important is that diversity is represented in complementarity-determining regions CDR1 and CDR2 in members of the different families as well as in members of the same VH family. These results suggest that an extensive repertoire of VH genes can contribute to antibody diversity in this lower vertebrate. Sequence comparisons indicate that one of the catfish VH families shares considerable structural similarity to several higher vertebrate VH gene families--a relationship which suggests that this VH family may be ancestral to some VH gene families of higher vertebrates. Characteristic of the genomic organization of higher vertebrate H chains, catfish appear to have different VH families wherein a VH gene likely undergoes functional recombination with putative DH gene segments and one of apparently several different JH segments. The recombined V region is expressed with the same C region gene. These combined results suggest that bony fishes are the earliest known phylogenetic representatives to have evolved extensive V region gene families.     

Neonatal and adult primary B cells use the same germ-line VH and V kappa genes in their (T,G)-A-L-specific repertoire

Although there is a nonrandom usage of VH gene families by primary B cells early in ontogeny, at issue is whether the preferential rearrangement of 3' germ-line VH genes, e.g., VH7183 and VHQ52 family genes, influences the neonatal B cell repertoire that can be expressed in response to Ag. In order to address this issue, and to determine whether neonatal B cells can use the same germ-line VH and V kappa genes as adult B cells in their primary response, we have analyzed at the molecular level the neonatal antibody response to (T,G)-A-L and compared it with the adult primary response. Among the TGB5 Id+, GT+ antibodies, which dominate the neonatal response to (T,G)-A-L, two VH gene families were used: J558 (high frequency) and 36-60 (low frequency). The majority of Id+ neonatal hybridomas used the same germ-line VH gene (H10, from the VHJ558 family), but with enormous diversity in the D region, and one of two germ-line V kappa 1 genes (V kappa 1A, V kappa 1C). These are the same germ-line V-genes used by most primary adult Id+ hybridomas, and the frequency of expression of this germ-line V-gene combination appears equivalent in the neonatal and adult primary repertoires. Therefore, it is clear from this study that as early as day 5, neonatal B cells can use the same germ-line V-genes as adult primary B cells in their Ag-specific repertoire.     

Analysis of VH gene replacement events in a B cell lymphoma

We have analyzed a series of recombinational events at the IgH chain locus of the B cell lymphoma, NFS-5. Each of these recombinational events results in the replacement of the VH gene segment of the rearranged H chain gene (VhDJh) with that of an upstream germline gene segment. Replacements on the productive and nonproductive alleles have been observed. In each case, the recombination occurs in close proximity to a highly conserved heptameric sequence (5'TACTGTG3') which is located at the 3' end of the VH coding region. In the two examples of recombination on the productive allele that have been analyzed, the initial VHQ52 gene is replaced by different VH7183 genes. On the non-productive allele, sequential replacement events have been analyzed: the initial VHQ52 rearrangement is first replaced by a closely related VHQ52 gene, followed by a second replacement using a VHQ52 pseudogene. Southern blot analysis using VH probes indicates that these recombinations may be accompanied by the deletion of germline VH genes belonging to both the VHQ52 and VH7183 families, suggesting that these gene families are interspersed in the NFS/N mouse.     

A Model for the Evolution of the Mammalian T-cell Receptor α/δ and μ Loci Based on Evidence from the Duckbill Platypus

The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called α, β, γ, and δ, which are expressed as either a αβ or γδ heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCRμ, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCRμ are also unclear, although it appears distantly related to TCRδ. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCRδ genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCRδ chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCRμ. They are expressed as part of TCRδ repertoire (VHδ) and similar to what has been found in frogs and birds. This, however, is the first time a VHδ has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCRμ. The current structure of TCRδ and TCRμ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCRδ genes, as well as translocations of TCRδ genes out of the TCRα/δ locus early in mammals, creating the TCRμ locus.     

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.     

Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly.

Immunoglobulin genes are assembled during lymphoid development by a series of site-specific rearrangements that are tightly regulated to ensure that functional antibodies are generated in B (but not T) cells and that a unique receptor is present on each cell. Because a common V(D)J recombinase comprising RAG1 and RAG2 proteins is used for both B- and T-cell antigen receptor assembly, lineage-specific rearrangement must be modulated through differential access to sites of recombination. We show here that the C-terminus of the RAG2 protein, although dispensable for the basic recombination reaction and for Ig heavy chain DH to JH joining, is essential for efficient VH to DJH rearrangement at the IgH locus. Thus, the RAG2 protein plays a dual role in V(D)J recombination, acting both in catalysis of the reaction and in governing access to particular loci.     

Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the kappa L chain and the TCR beta-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.     

VH replacement rescues progenitor B cells with two nonproductive VDJ alleles

Inaccurate VDJ rearrangements generate a large number of progenitor (pro)-B cells with two nonproductive IgH alleles. Such cells lack essential survival signals mediated by surface IgM heavy chain (muH chain) expression and are normally eliminated. However, secondary rearrangements of upstream VH gene segments into assembled VDJ exons have been described in mice transgenic for productive muH chains, a process known as VH replacement. If VH replacement was independent of muH chain signals, it could also modify nonproductive VDJ exons and thus rescue pro-B cells with unsuccessful rearrangements on both alleles. To test this hypothesis, we homologously replaced the JH cluster of a mouse with a nonproductive VDJ exon. Surprisingly, B cell development in IgHVDJ-/VDJ- mice was only slightly impaired and significant numbers of IgM-positive B cells were produced. DNA sequencing confirmed that all VDJ sequences from muH chain-positive B lymphoid cells were generated by VH replacement in a RAG-dependent manner. Another unique feature of our transgenic mice was the presence of IgH chains with unusually long CDR3-H regions. Such IgH chains were functional and only modestly counter-selected, arguing against a strict length constraint for CDR3-H regions. In conclusion, VH replacement can occur in the absence of a muH chain signal and provides a potential rescue mechanism for pro-B cells with two nonproductive IgH alleles.     

Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.     

A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).     

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.     

High frequency expression of S107 VH genes by peritoneal B cells of B10.H-2aH-4bP/WTS mice

Our previous analyses of peritoneal Ly-1 B cells indicate that a high percentage express VH genes of the VH11 and VH12 families, and that this bias is due to clonal selection. The antibodies encoded by these genes bind the same hapten, phosphatidyl choline (PtC). Twenty-one of 73 hybridomas generated from fusions with peritoneal Ly-1 and Ly-1 sister population B cells of B10.H-2aH-4bp/Wts mice produce anti-PtC specific antibodies. We show here that 19 of these express VH11 and VH12 family genes and two express VH36-60 family genes. To assess whether there is a bias in VH gene use among non-PtC-specific hybridomas we analyzed the remaining 52 hybridomas for VH family expression by using VH family-specific probes in an RNA dot blot assay and by Ig mRNA sequencing. We find a seven-fold increase in the expression of the VHS107 family genes, and only slight differences in the expression of VH genes of other families relative to splenic B cells. We attribute the increase in VHS107 gene expression to clonal selection inasmuch as five of the seven VHS107+ hybridomas express the same VH gene (V11) and VL association is nonrandom. The bias in VH gene use among the entire panel of 73 peritoneal hybridomas is to the extent that approximately one-third express one of three genes: the V11 gene of the S107 family, the CH34 gene of the VH11 family, and the VH12 family gene.