Antibodies generated from human immunoglobulin miniloci in transgenic mice.

One approach to the production of human monoclonal antibodies focusses on the creation of transgenic mice bearing human immunoglobulin gene miniloci. Whilst such loci undergo lymphoid-specific gene rearrangement, only a small proportion of mouse B cells express the human immunoglobulin chains; the miniloci thus contribute poorly to serum immunoglobulin. Attributing this poor performance to competition between the transgenic and endogenous immunoglobulin loci, we crossed mice bearing a human immunoglobulin heavy-chain (HulgH) minilocus with animals that had been rendered B cell-deficient by disruption of their endogenous heavy-chain locus. The results were dramatic: the human minilocus rescued B cell differentiation such that effectively all B cells now expressed human mu chains. The concentration of antibody in the mouse serum recognised by anti-human mu increased to a concentration about one sixth that in human serum. The HulgH antibodies are heterogenous with diversity being generated by both combinatorial and junctional processes. Following antigen challenge, specific antibody is elicited but at low titre.     

Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D_H) and joining (J_H) gene segments were methylated, DJ_H junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.     

An enhancer at the 3'' end of the mouse immunoglobulin heavy chain locus.

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.     

Allele Distribution at the HVR-IgH Hypervariable Locus in Unrelated Individuals Living in the Urals, Siberia, and Northern Kazakhstan

Allelic polymorphism at a hypervariable locus mapped to the 5"-region of the gene cluster encoding J_H segments of human immunoglobulin heavy chain H (IgH-VNTR) was typed in 462 unrelated individuals living in the Urals, Siberia, and Northern Kazakhstan. Molecular characteristics of the three previously unknown IgH-VNTR alleles are presented. The observed genotype frequencies were concordant with those expected under the Hardy–Weinberg distribution. The data obtained can be used in the work of regional forensic laboratories.     

Allelic polymorphism of mouse Igh-J locus,which encodes immunoglobulin heavy chain joining (JH) segments

The mouse genome contains four functional J _H genes, which encode immunoglobulin heavy chain joining segments. The J _H gene cluster is located a few kilobases 5 from the constant region genes (C genes) on chromosome 12. The polymerase chain reaction (PCR)-technique was used to amplify DNA stretches from mouse genome of approximately 1 340 nucleotides in length containing all four J _H genes (Igh-J locus). PCR products were directly used as templates in Sanger's dideoxy-sequencing, and sequences were determined. Twelve inbred mouse strains belonging to ten different Igh-C haplotypes were studied. The strains were: BALB/c, C58/J, RIII, DBA/2, CE, RF, CBA, NZB/J, AKR, C57BL/10, SJL, and A/J. Five allelic forms of the Igh-J locus were found among these strains. The A/J mouse has an allele (e) which differs from the BALB/c allele (a) by 15 nucleotides. C57BL and SJL have the allele (b) with eight differences from BALB/c. The CBA allele (j) has two differences, and the CE allele (f) has a single nucleotide difference compared with the BALB/c sequence. Based on the J _H , variable (V) and constant (C) region sequences we conclude that independent reshuffling of V _H ,J _H , and C _H gene clusters occurred during the evolution of Mus musculus.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X63146-X63175.     

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.     

Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells

We have produced a series of hybrid IgG₁-IgG_2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differnces between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse γ¹ and γ^2a gene segments. Crossovers occured through out these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H₂L₂ tetramers and properly glycosylated. In addition, all 11 immunoglbulins have identical antigen combining sites specific for the fluorescent hapten ε-dansyll-L-lysine. Protein A binding was used as probe of the structural integrity of the Fc portion of the variant antibodies. Differeneces in protein A binding between IgG₁ and IgG_2a appear to be due to amino acid differances at postions 252 (Thr→Met) and 254 (Thr→Ser) of the heavy chain (EU numbering).     

Contribution of VH Replacement Products in Mouse Antibody Repertoire

V_H replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged V_H gene and the 23-bp RSS from an upstream unrearranged V_H gene. Due to the location of the cRSS, V_H replacement leaves a short stretch of nucleotides from the previously rearranged V_H gene at the newly formed V-D junction, which can be used as a marker to identify V_H replacement products. To determine the contribution of V_H replacement products to mouse antibody repertoire, we developed a Java-based V_H Replacement Footprint Analyzer (V_HRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify V_H replacement products. The overall frequency of V_H replacement products in these IgH genes is 5.29% based on the identification of pentameric V_H replacement footprints at their V-D junctions. The identified V_H replacement products are distributed similarly in IgH genes using most families of V_H genes, although different families of V_H genes are used differentially. The frequencies of V_H replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified V_H replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of V_H replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.     

Genetic stability of gene-targeted immunoglobulin loci. II. Influence of the cell line and the vector linearization site

The site-specific integration of exogenous gene fragments by homologous recombination provides a convenient method for altering the immunoglobulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybridoma cells we compared the recombination frequencies of vectors that could be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombination was observed, irrespective of the location of the site at which the vector was cleaved. Integration events involving the human IgG1 vectors were lost at high frequency due to secondary vector excision, so that all stable recombinations were found to be replacement events. Replacement recombination of an integration vector involves an illegitimate crossover at least at the 3′ side and sometimes gives rise to deletion of the C_H1 domain. However, a homologous event at the 3′ side is more efficient than an illegitimate one, so that a homology that is distributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total length. The position of the linearization site in the vector markedly influenced the targeting efficiency, but surprisingly, whether a double-strand break in the homology or in the heterology region more efficiently promoted integration was dependent on the cell line. In all cells, however, cleavage of the vector outside the homology region favoured stable replacements with a bias against C_H1-truncated clones. We further show that the frequency of replacements induced by integration vectors is not correlated to the homology length and cannot be increased by irradiation of the cells. Our findings indicate that for targeting the IgH locus other mechanisms might be involved than at other loci. Received: 20 January 1997 / Accepted: 9 June 1997     

Genetic stability of gene-targeted immunoglobulin loci. II. Influence of the cell line and the vector linearization site

The site-specific integration of exogenous gene fragments by homologous recombination provides a convenient method for altering the immunoglobulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybridoma cells we compared the recombination frequencies of vectors that could be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombination was observed, irrespective of the location of the site at which the vector was cleaved. Integration events involving the human IgG1 vectors were lost at high frequency due to secondary vector excision, so that all stable recombinations were found to be replacement events. Replacement recombination of an integration vector involves an illegitimate crossover at least at the 3′ side and sometimes gives rise to deletion of the C_H1 domain. However, a homologous event at the 3′ side is more efficient than an illegitimate one, so that a homology that is distributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total length. The position of the linearization site in the vector markedly influenced the targeting efficiency, but surprisingly, whether a double-strand break in the homology or in the heterology region more efficiently promoted integration was dependent on the cell line. In all cells, however, cleavage of the vector outside the homology region favoured stable replacements with a bias against C_H1-truncated clones. We further show that the frequency of replacements induced by integration vectors is not correlated to the homology length and cannot be increased by irradiation of the cells. Our findings indicate that for targeting the IgH locus other mechanisms might be involved than at other loci.     

A single VH gene segment encodes the immune response to phosphorylcholine: Somatic mutation is correlated with the class of the antibody

The immune response in BALB/c mice to phosphorylcholine is highly restricted in its heterogeneity. Of the 19 immunoglobulins binding phosphorylcholine for which complete V_H-segment amino acid sequences have been determined, 10 employ a single sequence, denoted T15 after the prototype V_H sequence of this group of antibodies. The remaining 9 of these V_H segments are variants differing by 1 to 8 residues from the T15 sequence. Using a cloned V_H cDNA probe complementary to the T15 sequence, we isolated from a mouse sperm genomic library clones corresponding to four V_H gene segments that by DNA sequence analysis are >85% homologous to one another. These four V_H gene segments have been denoted the T15 V_H gene family. These V_H gene segments are most, if not all, of the germline V_H gene segments that could encode the V_H sequences of antibodies that bind phosphorylcholine. One of these four genes contains the T15-V_H-coding sequence. When the T15-family V_H gene segments were compared with the complete V_H protein sequences of 19 hybridoma and myeloma immunoglobulins that bind phosphorylcholine, several striking conclusions could be drawn. First, all of these V_H regions must have arisen from the germline T15 V_H gene segment. Thus virtually the entire immune response to phosphorylcholine is derived from a single V_H-coding sequence. Nine of the 19 V_H regions were variants differing from the T15-V_H-coding sequence and, accordingly, must have arisen by a mechanism of somatic diversification. Second, the variants appear to be generated by a somatic mutation mechanism. They cannot be explained by recombination or gene conversion among members of the T15 gene family. Third, somatic mutation is correlated with the class of the antibody. All of the somatic variation is found in the V_H regions derived from antibodies of the IgA and IgG classes. The IgM molecules express the germline T15 V_H gene segment exclusively.     

Chimeric IgH-TCRα/δ translocations in T lymphocytes mediated by RAG

Translocations involving the T cell receptor alpha/delta (TCRα/δ) chain locus, which bring oncogenes in the proximity of the TCRα enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRα/δ locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRα/δ associated DNA cleavage, but the molecular mechanism that leads to generation of the "oncogene partner" DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRα/δ fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRα/δ as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs.     

The chromosome breakpoint at 14q32 in an ataxia telangiectasia t(14;14) T cell clone is different from the 14q32 breakpoint in Burkitts and an inv(14) T cell lymphoma

Summary The T cell receptor chain gene locus and the immunoglobulin heavy chain gene locus (IgH) have previously been mapped to the q11 and q32 positions respectively of the human chromosome 14. Both of these sites are also common breakpoints in lymphocytes from ataxia telangiectasia (A-T) patients. Using in situ hybridisation we show that the 14q32 breakpoint in an A-t non-leukaemic T cell clone with t(14;14) translocation, lies outside the IgH locus and proximal to it with respect to the centromere. The 14q11-14qter segment of the homologous chromosome 14 carrying the constant gene region of the chain locus is translocated to this 14q32 position.     

Immunoglobulin JH rearrangement in a T-cell line reflects fusion to the D H locus at a sequence lacking the nonamer recognition signal

Rearrangements of the immunoglobulin heavy chain joining region (J _H ) genes occur in some T lymphocytes, probably because the mechanism for assembly of T-cell receptor encoded genes is very similar to that for immunoglobulins. Two such rearrangements described previously represented proper fusion of a D _H and J _H gene. We have cloned and analyzed the J_H rearrangements found on both alleles in the T lymphoma ST4. One represents conventional recombination between a member of the SP2 D _H family and J _H3 . On the other allele, J_H4 has recombined with a sequence within the D _H locus but not at a D _H gene. This recombination involved the heptamer but not the nonamer of the bipartite recognition signal required for D _H -J _H joining. This result suggests that the heptamer may be the primary determinant of the specificity in V-gene assembly and that the D _H locus as a whole may be a preferred target for recombination.     

Nuclear factors binding to the human immunoglobulin heavy-chain gene enhancer.

The human immunoglobulin heavy chain (IgH) gene contains at least two tissue-specific regulatory regions, which are similar to the mouse IgH gene. One is the J-C enhancer and another is located in the 5' promoter region. Using an electrophoretic mobility shift assay and DNase I footprint, we have examined the interaction of factors in B cell nuclear extracts with the two regulatory regions of the human IgH gene. We have identified a nuclear factor in mouse B cell nuclear extracts which bound to specific sequence in the human IgH enhancer. This factor is apparently not present in mouse fibroblast nuclear extracts. We also found factor(s) which bound to the highly conserved octanucleotide sequence within the human IgH enhancer and 5' promoter regions.     

Human Immunoglobulin Repertoires against Tetanus Toxoid Contain a Large and Diverse Fraction of High-Affinity Promiscuous VH Genes

To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V_H) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V_L) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V_H regions in this V_L-restricted panel with a previously published repertoire of anti-TT V_H regions with cognate V_H-V_L pairing showed a very similar distribution of V_H, D_H and J_H gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V_L anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V_L anti-TT V_H repertoire was combined with a collection of naïve V_L regions and again selected for binding to TT, many of the V_H genes were recovered in combination with a diversity of V_L regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V_H combined with 15 diverse V_L chains were determined and found to be identical to each other and to the original isolate restricted to a single-V_L chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V_H regions from an immunized repertoire have promiscuous features. These V_H regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V_H region diversity and affinity of a natural repertoire.