Sequences of the joining region genes for immunoglobulin heavy chains and their role in generation of antibody diversity

To assess the contribution to immunoglobulin heavy chain diversity made by recombination between variable region (VH) genes and joining region (JH) genes, we have determined the sequence of about 2000 nucleotides spanning the rearranged JH gene cluster associated with the VH gene expressed in plasmacytoma HPC76. The active VH76 gene has recombined with the second germ-line JH gene. The region we have studied contains two other JH genes, designated JH3 and JH4. No other JH gene was found within the region 1000 nucleotides downstream from JH4. Between JH3 and JH4 there is a pseudo-JH sequence with substantial homology to the authentic JH genes. The four JH genes whose sequences now are known can account for all known JH amino acid sequences. The JH genes are more divergent than the Jk genes and vary in length, encoding either 15 or 17 amino acid residues. Because JH regions comprise part of the third hypervariable region (HV3), combinatorial VH--JH joining substantially augments VH diversity. Moreover, a VH gene can recombine with each JH gene at several positions, and either one or two germ-line JH codons can be excised. This JH truncation markedly reduces the length of HV3 and hence must alter antigen-binding specificity. We have also determined the sequence of the JH4 region in two different gamma 2a mRNAs and have found that each has suffered a point mutation (aspartate to asparagine) which would alter the charge of the antigen-binding site.     

Preferential use of the VH4 Ig gene family by diffuse large-cell lymphoma

Ig heavy chain variable region (VH) genes expressed by human diffuse large-cell lymphoma (DLC) and follicular lymphoma (FL) were identified and analyzed with respect to germline gene families. In 67 cases of FL, VH region genes were expressed in a pattern similar to that of normal B cells, with a predominance of the large VH3 gene family being used. In contrast, of the 17 cases of DLC, there was an extremely biased use of VH genes. Of these DLC tumors, 88% expressed genes from the small VH4 gene family; and even among these tumors, there was a limited use of genes, with 11 cases producing Igs derived from the VH4.21 germline gene. Although most of the VH genes expressed by DLC tumor cells contained mutations with respect to their germline counterparts, almost all of these mutations occurred before the clonal expansion of the tumor. This contrasts with our previous findings of ongoing mutations in FL and represents a fundamental difference between these two malignancies. This preferential gene use implies an important role for the VH4 gene family, and specifically for VH4.21, in the genesis of DLC.     

Somatic rearrangements forming active immunoglobulin mu genes in B and T lymphoid cell lines.

We have cloned an active gene for an immunoglobulin mu heavy (H) chain, bearing the variable (VH), joining (JH), and constant (C mu) sequences expressed in the IgM-secreting mouse plasmacytoma HPC-76. The mu gene was formed by somatic recombination between a VH gene and one of several JH genes, which are located about 7.7 kilobase pairs from the C mu gene in embryo DNA. The JH-C mu intervening sequence has suffered a deletion of about 2.7 kilobase pairs in HPC-76. Because the delection encompasses sequences required to switch an expressed VH-JH gene from C mu to another CH gene, it may represent a mechanism for "freezing" a lymphocyte clone at the stage of IgM expression. For the second (inactive) C mu allele in HPC-76, the entire joining and switch regions have been deleted; functional inactivation of one allele may thus represent one mechanism by which a lymphocyte clone restricts expression to a single allele (allelic exclusion). Probes generated from the cloned mu gene allowed examination of the JH locus in B, Abelson "pre-B," and T lymphoma cell lines and a myeloid line, all of which cotain RNA species bearing C mu sequences. The B and pre-B lines exhibited recombination within both alleles of the JH locus, suggesting that both alleles may be expressed in some cells. The absence of the JH gene 5' to the recombination sites favors a deletion mechanism for VH-JH joining. Recombination within the JH locus was also detected in two out of four T lymphoma lines, but not in the myeloid line. This indicates that the mechanism by which B cells generate immunoglobulin diversity is operational in some T cells. Lines that synthesize mu RNA without JH rearrangement may have activated the C mu gene directly or have undergone recombination at a more distant locus.     

Restricted diversity of the variable region nucleotide sequences of the heavy and light chains of a human rheumatoid factor

The complete nucleotide sequences of the variable region genes of the heavy and light polypeptide chains of a human monoclonal rheumatoid factor (RF) produced from a human-mouse heterohybridoma were determined. The antibody, designated YES8c, contained V kappa III, J kappa 2, VH1, JH4, and a D gene segment of 9 amino acids. The nucleotide sequences and the deduced amino acid sequences of the light chain variable region were remarkably homologous (97-98%) to previously described RF of the Wa idiotypic family (PAY, GLO, CUR, FLO, and GAR) and to that of a V kappa III germline gene (Humkv325). The YES8c heavy chain variable region gene was most closely related to the VH1 gene of the restricted human fetal repertoire, designated 51p1, and also to 3 rearranged VH1 genes that were recently isolated from patients with chronic lymphocytic leukemia. These results suggest that variable region genes of RFs are highly conserved and that YES8c VH, as well as V kappa, may be identical to heavy and light chains expressed during early B cell development.     

V gene usage by seven hybrids derived from CD5+ B-cell chronic lymphocytic leukemia and displaying autoantibody activity

We report here the complete heavy and light chain variable region sequences of seven heterohybridomas derived from CD5+ chronic lymphocytic leukemia (CLL) B lymphocytes and displaying natural autoantibody activity. The three hybrids displaying a polyreactive pattern of binding used VH4 family members, ie, the VH4-18 gene in germinal configuration in two cases and a VH4 gene with 90% homology with VH4-21 for the third one. A hybrid expressing anti-Sm activity used a VH3 family member with 95.26% homology with the 30P1 gene. The three hybrids exclusively displaying rheumatoid factor activity expressed VH1 family genes: 51P1 gene for two (in germinal configuration in one, and with 93.2% homology in the other), whereas the third one used the V1-3b gene (98.8% homology). Definitive homology with known germline D segments was found for four of the seven hybrids (DN2 in 3 and DLR4 in 1) and JH use appeared to be random. The three hybrids displaying polyreactive activity expressed V kappa I, V lambda III, and V lambda II genes, all in germinal configuration. Among the three hybrids with rheumatoid factor activity, two used the same V kappa II gene with, respectively, 98% and 96% homology with a gene previously described; the third used a V lambda I gene in germinal configuration. Finally, the clone with anti-Sm activity used a V lambda III gene having 97% homology with a germinal gene. Overall, these results attempt to establish the relationship between frequent self- reactivity observed in CD5+ B-CLL and V gene usage. For VH genes, they confirm overexpression of the 51P1 gene in B-CLL and suggest nonstochastic use of two VH4 genes (4-21 and 4-18). For VL genes, available information is too scarce to lead to firm conclusions.     

Two acquired immunodeficiency syndrome-associated Burkitt's lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments

We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4-21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4-21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4-21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4-21 gene in the human population, the nature of the nucleotide differences in the expressed VH4-21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4-21 gene. Thus, the expression of VH4-21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.     

Physical linkage of a human immunoglobulin heavy chain variable region gene segment to diversity and joining region elements.

Antibody genes are assembled from a series of germ-line gene segments that are juxtaposed during the maturation of B lymphocytes. Although diversification of the adult antibody repertoire results in large part from the combinatorial joining of these gene segments, a restricted set of antibody heavy chain variable (VH), diversity (DH), and joining (JH) region gene segments appears preferentially in the human fetal repertoire. We report here that one of these early-expressed VH elements (termed VH6) is the most 3' VH gene segment, positioned 77 kilobases on the 5' side of the JH locus and immediately adjacent to a set of previously described DH sequences. In addition to providing a physical map linking human VH, DH, and JH elements, these results support the view that the programmed development of the antibody VH repertoire is determined in part by the chromosomal position of these gene segments.     

The genetic variability of the VH genes in follicular lymphoma: the impact of the hypermutation mechanism

Follicular lymphoma (FL) cells have inherited an activated hypermutation mechanism from their origin of germinal centre B cells. Based on today's knowledge of the intrinsic properties related to this mechanism and the VH base composition, reconsideration of previous reports should be made on a broader range of samples. The present study examined the mutation pattern of the VH genes expressed by 55 cases of FL. FL VH genes showed evidence of antigenic selection in 30% of cases with 88% carrying a functional sIg and 78.2% showing intraclonal variation. VH family and gene segment utilization was found to be roughly similar to that of normal B lymphocytes. FL VH genes revealed extensive variations. 17% of the VH exons harboured a total of five deletions, three duplications and two insertions as compared to the most homologous germline counterpart. The VH genes of one tumour displayed three populations with varying CDR3 length at diagnosis. At relapse, emergence of a differently mutated gene, additional mutations reminiscent of ongoing mutations or no variation was prominent. From this study the heterogeneity of FLs is well established and ongoing mutations are seen in the scope of the activated status of the hypermutation mechanism rather than antigen-stimulated tumour growth.     

Preferential expression of variable region heavy chain gene segments by predominant 2,4-dinitrophenyl-specific BALB/c neonatal antibody clonotypes.

The B-cell repertoire in neonatal mice contains predominant clonotypes that are reproducibly expressed at particular times after birth. We have isolated and sequenced heavy and light chain cDNA clones from three 2,4-dinitrophenyl-specific neonatal hybridomas. Two of these hybridomas (TF2-36 and TF5-139) express idiotypes (Ids) that predominate during the first days after birth, and the third hybridoma (TF2-76) expresses an Id that predominates during the second week after birth. The heavy (H) chain variable (V) region of the TF2-76 hybridoma protein is encoded by a member of the 7183 VH family, one of eight families of murine VH genes that have been defined by Brodeur and Riblet [Brodeur, P. H. & Riblet, R. (1984) Eur. J. Immunol. 14, 922-930]. Members of this family have been found to undergo a disproportionately high frequency of rearrangement in fetal and neonatal liver pre-B-cells. Because the 7183 VH family is located close to the H chain joining (J) region gene segments, JH, other workers have proposed that VH rearrangement frequency is related to distance from the JH segments. However, the two earlier-appearing predominant clonotypes expressed by TF2-36 and TF5-139 hybridoma proteins utilize a member of the 36-60 VH family, probably VH 1210.7, which is located distal to the JH gene segments on chromosome 12. Since 20-30% of day 3 dinitrophenyl-specific B cells express either the Id(TF2-36) or the Id(TF5-139), the VH 1210.7 gene must be utilized at high frequency early in development. These results indicate that the utilization of rearranged VH segments is strongly influenced by factors other than distance from JH.     

Chromosomal organization of the heavy chain variable region gene segments comprising the human fetal antibody repertoire.

The adult repertoire of antibody specificities is acquired in a developmentally programmed fashion that, in mouse and man, parallels the ordered rearrangement of a limited number of germ-line heavy chain variable region (VH) gene segments during development. It has been hypothesized that this developmental bias is a consequence of gene organization. In the mouse, rearrangement of VH gene segments proximal to the heavy chain joining region (JH) locus precedes rearrangement of genes located more distal to the JH locus. Similarly, in man, two VH elements located proximal to JH are expressed during fetal development. To test further this hypothesis in man, we have determined in a single individual the positions of an additional eight distinct VH elements known to comprise a significant fraction of the human developmental repertoire. These developmentally expressed VH elements were found to be dispersed over a region of 890 kilobases of the VH locus and were interspersed with other VH elements that are not known to be developmentally expressed. Thus, the ordered developmental expression of VH gene segments in man must involve mechanisms beyond physical proximity to the JH locus. Further, these results support the notion that fetal expression of VH gene segments is a regulated process and suggest that this regulation is important in the acquisition of immunocompetence.     

Morphologic transformation of follicular lymphoma is associated with somatic mutation of the translocated Bcl-2 gene

Follicular lymphoma (FL) is a low-grade B-cell non-Hodgkin's lymphoma (NHL) that frequently transforms into diffuse aggressive NHL. The majority of FLs display a t(14; 18) translocation that places the bcl-2 gene into juxtaposition with the lg heavy-chain (H) gene locus. Morphologically transformed malignant FL cells retain their t(14;18) translocation and may acquire additional genetic abnormalities. We analyzed serial biopsy specimens from eight patients with FL for secondary alterations of the rearranged bcl-2 gene in the breakpoint and open reading frame (ORF) regions. Two cases of FL showed no histologic alteration in the second biopsy, and six cases of FL showed morphologic transformation to diffuse large-cell lymphoma (DLL) in the second biopsy. Polymerase chain reaction (PCR) amplification, cloning, and sequencing of the junctional region of the hybrid bcl-2/IgH genes showed identical nucleotide sequences in multiple biopsy specimens of FL that did not show morphologic transformation. In patients in whom FL cells underwent morphologic transformation, FL and autologous DLL cells displayed identical bcl-2/IgH gene nucleotide sequences in five cases and different sequences in one case. In the case for which FL and DLL cells showed different bcl-2/IgH junctional sequences, DLL cells incorporated larger bcl-2 and Ig-joining (JH) gene fragments than the corresponding FL cells, suggesting that DLL clones developed by a distinct t(14; 18) translocation rather than by alteration of the hybrid bcl-2/IgH gene detected in the FL cells. In all eight cases, neither FL nor DLL cells showed alterations of bcl-2 gene sequences in the breakpoint region, suggesting high conservation of the bcl-2 gene during both t(14; 18) translocation and morphologic transformation of the FL cells. PCR single-strand conformation polymorphism (SSCP) and sequence analyses were performed for identification of structural alterations of the bcl-2 gene in the ORF region corresponding to the 239-amino acid p26-bcl-2 alpha protein. A total of 11 point mutations of the ORF were detected in DLL cells of three transformed NHLs, but no alteration of the ORF was detected in FL cells. Four of 11 mutations, at positions 29, 46, 59, and 106, yielded amino acid replacements. These findings demonstrate that FL and DLL cells may be clonally related or unrelated. They also show that transformation of FL cells may be associated with somatic point mutations of the bcl-2 proto- oncogene ORF sequence resulting in alteration of the p26-bcl-2 alpha gene product.     

Evolutionary aspects of immunoglobulin heavy chain variable region (VH) gene subgroups.

We isolated and determined the sequences of two human germ-line heavy chain variable region (VH) genes and compared them with mouse VH genes. The results show that the human VHI subgroup is evolutionarily related to the mouse VHII subgroup. Evolutionary preservation of homologies in VH genes of the same subgroup includes not only the coding region but also intron size and homology in noncoding regions. This suggests that a VH gene subgroup constitutes a multigene family that undergoes concerted evolution. The homology between genes of the same subgroup in different species is greater than that between genes of different subgroups within a species. One of the VHII genes contains, in complementarity-determining region 2 (CDR2), a 13-base-pair previously shown to be in CDR2 of a VHIII gene and in a heavy chain diversity region gene, DH [Wu, T. T. & Kabat, E. A. (1982) Proc. Natl. Acad. Sci. USA 79, 5031-5032], suggesting the insertion of diversity region gene sequences into the VH gene. One of the human VH genes is a pseudogene because of a terminator, which, together with our previous results, shows that the VH gene repertoire contains 40% pseudogenes. In one of the VH genes, direct and inverted repeats at both 5' and 3' ends of the gene suggest a potential transposable element that encompasses the entire VH gene. It is possible that such a structure may facilitate saltatory replication and rapid expansion of VH gene families.     

Expression and rearrangement of homologous immunoglobulin VH genes in two mouse strains.

A family of murine anti-p-azophenylarsonate (Ars) antibodies share a variable (V) region serologically defined marker, the 36-60 idiotype (Id36-60). Most mouse strains possess five genes highly homologous to the gene encoding the heavy (H) chain V region of antibodies bearing Id36-60 (VH36-60); however, only one of these genes is ever utilized by hybridomas whose antibodies bind Ars and bear Id36-60. The relevant VH genes were cloned from A/J and BALB/c mouse DNA libraries. Their DNA sequences were found to differ at only two positions. Southern blot analysis, protein sequence determination, and nucleic acid sequence determination indicate that the above hybridomas utilize the same joining (JH3), diversity (D), and VH gene segments regardless of BALB/c or A/J strain origin. Despite this virtual identity, BALB/c and A/J mouse strains express quite different serum levels of Id36-60-bearing antibodies when immunized with Ars. The basis of this regulatory process is discussed.     

VH mutation status and VDJ rearrangement structure in mantle cell lymphoma: correlation with genomic aberrations,clinical characteristics,and outcome

Immunoglobulin variable heavy chain gene (VH) mutation status and VDJ rearrangement structure were analyzed in 141 patients with mantle cell lymphoma (MCL) and correlated with biologic and clinical characteristics; 29% of the MCLs displayed mutated VH using a 98% germline homology cutoff. Striking differences occurred in the VH mutation subgroups with respect to the use of specific V genes. Rearrangements involving V4-34 and V3-21 were almost exclusively unmutated, whereas rearrangements using V4-59 and V3-23 were typically mutated. Significant association occurred between mutated VH with shorter CDR3 lengths and the use of JH4b. V3-21 and V4-59 were involved in highly characteristic rearrangements, implying that antigen specificity might have been involved in MCL development. There was no evidence for isotype switch recombination or Bcl-6 expression in any MCL. ZAP70 expression was not different in VH-mutated or -unmutated MCL. Although the deletions 11q- and 17p- showed a balanced distribution, an overrepresentation was observed for trisomies +3q, +8q, and tetraploidy in the VH-unmutated subgroup and +12q in the VH-mutated subgroup. Clinically, mutated VH was associated with a higher rate of complete remission, but there was no correlation between VH mutation status and other clinical characteristics or overall survival.     

Immunoglobulin heavy chain variable region gene evolution: structure and family relationships of two genes and a pseudogene in a teleost fish.

Nucleotide sequences for two immunoglobulin heavy chain variable region (VH) genes and one pseudogene in the goldfish (Carassius auratus) and the family relationships and distribution of these genes in individual fish are presented. Comparison of the nucleotide and inferred amino acid sequences of goldfish and other vertebrate VH genes indicates that goldfish VH genes show the major VH gene regulatory and structural features (5'-putative promoter region, split hydrophobic leader, three framework and two complementarity-determining regions, and 3'-recombination signals for VH to diversity region joining) and that goldfish VH genes are not more closely related to one another than they are to VH genes of evolutionarily distant vertebrates such as the mammals. Goldfish VH genes appear to exist in distinct families, and individual goldfish can carry from none to apparently greater than 15 genes of a given family. These results suggest that whereas the basic structure of VH genes has been conserved in evolution, there may be substantial variation in the nature and population distribution of VH gene families in the vertebrates.     

VH and VL gene analysis in sporadic Burkitt's lymphoma shows somatic hypermutation, intraclonal heterogeneity, and a role for antigen selection

Tumor cell lines and one tumor biopsy from seven cases of Epstein-Barr virus (EBV) genome-negative sporadic Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig variable region genes. The VH genes were derived from the VH 3 (one) and VH4 (six) families and both the IgM-positive (six) and the IgA-positive (one) were all mutated from their germline counterparts. The VL genes were derived from V kappa 1 (one), V kappa 3 (one), V lambda 1 (four), and V lambda 2 (one) families and were also somatically hypermutated. Biopsy material from one of the IgM-positive cases showed VH and VL sequences that matched the derived cell line, with additional intraclonal sequence heterogeneity, indicating that the tumor cells had undergone posttranformation somatic mutation. Mutational patterns in V(H) genes did not show a conventional role for antigen in selecting tumor cell sequences. In contrast, patterns in VL sequences were consistent with a role for antigen in five of seven cases. The pattern of extensive scattered somatic hypermutation and intraclonal variation is similar to that in VH sequences of EBV genome-positive endemic BL, although the degree of mutational activity is less. These common features indicate that B cells involved in the two variants of BL may share a common clonal history.     

Translocation of immunoglobulin VH genes in Burkitt lymphoma.

We have produced cell hybrids between mouse myeloma cells, which do not produce immunoglobulin chains, and Burkitt lymphoma cells (Daudi), which express surface IgM. Daudi Cells carry a reciprocal chromosome translocation between chromosomes 8 and 14, described as (8;14)(q24;q32). The hybrids were studied for the expression of human immunoglobulin chains and human isozyme markers, for the presence of human chromosomes, and for the presence of the human genes for heavy chain variable regions (VH) and mu and gamma chain constant (C) regions. The results indicate that the expressed mu chain gene is on normal chromosome 14 in Daudi cells. We have also determined that the chromosome 14 involved in the translocation (14q+) carries the gene for C mu and C gamma 1-4 and probably several genes for the variable region (V). Certain hybrids had lost both the chromosomes 14 but had retained the abnormal chromosome 8 (8q-) that carries the terminal end of the long arm of chromosome 14. These hybrids were studied for the presence of human VH, C mu,, and C gamma DNA sequences, and the results indicated that the hybrid cells with the 8q- chromosome contained VH genes that not C genes. Therefore, we conclude that, in the Daudi Burkitt lymphoma, the break in chromosome 14 occurred within the chromosome segment containing V region genes. As a result of the translocation some of these VH genes became associated with chromosome 8. It is possible that the expression of malignancy in Burkitt lymphoma is caused by immunoglobulin V region gene translocation resulting in activation of a gene on the long arm of human chromosome 8.