Rearrangement of only one human IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses in transgenic mice

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.     

Nucleotide sequence analysis of a human monoclonal antibody TONO-1 with cytotoxic potential for T-leukemia/lymphoma cells

A human monoclonal antibody (HuMab) TONO-1 (IgM, lambda) recognizes cell surface antigens associated primarily with human T-leukemia/lymphoma cells. In this study, we investigated the reactivity against T-leukemia/lymphoma cells in detail, cytotoxic potential and primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMab TONO-1. Expression of the molecules (TONO-1 Ags) detected by a HuMab TONO-1 was significantly heterogeneous even in the same T-leukemia/lymphoma cell lines HPB-MLT and MOLT-4F. The flow cytometric curves showed an unusual broad-based spread of fluorescence intensity. HuMab TONO-1 was shown to have the ability to kill the T-leukernia/lymphoma cells efficiently in the presence of rabbit complements. However, HuMab TONO-1 did not demonstrate significant antibody-dependent cellular cytotoxic activity. Furthermore, HuMab TONO-1 heavy and light chain variable regions were cloned, sequenced and analyzed. HuMab TONO-1 uses a V(H) gene member of the V(H)IV gene family V(H)71-4, and is productively rearranged with the germ line D(H) gene D(XP')1, and the germ line J(H)5 gene with multiple somatic mutations. HuMab TONO-1 Vlambda belongs to the lambda light chain variable subgroup I family and is derived from the Vlambdalc germ line gene Humlv1042, and germ line gene Jlambda1 without somatic mutations. The results reveal that the production of HuMab TONO-1, with cytotoxic potential for human T-leukemia/lymphoma cells, is achieved by rearrangement of the V(H)71-4/Humlv1042 germ line variable region gene combination, that is associated with the autoimmune repertoire.     

Immunoglobulin heavy and light chain gene sequences of a human CD5 positive immunocytoma and sequences of four novel VHIII germline genes.

To analyse the V genes expressed by an IgM lambda CD5-positive immunocytoma heavy and light chain V region genes were cloned and sequenced. The heavy chain is composed of a previously undescribed VHIII gene joined to an unknown D gene and to JH4. The light chain V region is composed of a V lambda II gene rearranged to J lambda 1. In an attempt to clone the germline counterpart of the VHIII gene expressed in the immunocytoma PCR amplifications of genomic DNA were carried out and four previously unknown VHIII genes were identified. As several independent clones for the heavy and light chain V region genes were sequenced the rate of somatic mutation of the V genes was calculated to be below 2 x 10(-5)/bp/cell division.     

Molecular and cellular basis of the altered immune response against arsonate in irradiated A/J mice autologously reconstituted.

The humoral immune response to arsonate (Ars) in normal A/J mice is dominated in the late primary and particularly in the secondary response by a recurrent and dominant idiotype (CRIA) which is encoded by a single canonical combination of the variable gene segments: VHidcr11-DFL16.1-JH2 and Vkappa10-Jkappa1. Accumulation of somatic mutations within cells expressing this canonical combination or some less frequent Ig rearrangements results in the generation of high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Ars-keyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with Ars-KLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow.     

By-products of immunoglobulin somatic hypermutation

The antigen receptor loci are the only loci in humans to undergo programmed somatic gene modification. Although aberrant V(D)J integration and class switch recombination can both give rise to chromosomal translocations, a role for somatic hypermutation in such genomic rearrangements has been suggested but is less clearly established. To characterize the types of by-products of somatic hypermutation, we analyzed aberrant rearrangements involving the immunoglobulin loci in a human B-cell line (Ramos) that performs Ig V gene hypermutation constitutively during culture. Single-nucleotide substitutions account for 95% of the mutational events in the VH gene, with small deletions and duplications accounting for most of the remaining mutations. However, larger genetic alterations can be detected at low frequency, accounting for 0.5% of VH-inactivating mutations. Examples include a large (13 kb) deletion, which entirely removes the expressed VH gene; a 3-kb deletion extending from the functional VHDJH into an upstream VH (generating a hybrid VHDJH gene reminiscent of VH replacement); and an ectopic insertion into the VH from chromosome 1. The results support the proposal that aberrant antibody hypermutation can lead to gross genomic alterations but indicate that such events are rare.     

Diversity of novel recombining elements suggests developmentally programmed expression of the T cell receptor alpha/delta locus.

During fetal ontogeny, the first wave of gamma delta T lymphocytes appears in the thymus at day 14 of gestation assembling predominantly T cell receptors (TcR) with V gamma 3 and V delta 1. To identify V delta gene segments that are transcribed at day 16, subsequent to the first wave of V delta 1 expression, delta chain cDNA was amplified by the anchored polymerase chain reaction with single-sided specificity for C delta. Unexpectedly, most of the cDNA clones do not contain V gene segments. In some cDNA clones an alternative splice from the leader exon to the C delta exon has deleted the whole variable region exon. In other cDNA clones, multiple non-V-like elements are juxtaposed to the D delta 2 and J delta 1 gene segments. A large number of these diverse elements appear to be rearranged in fetal thymocytes, bringing V alpha gene segments located upstream of the recombining element into proximity to the J alpha locus. It is proposed that these rearrangements make irreversible the commitment to the TcR alpha beta lineage and determine a programmed read out of different clusters of V alpha gene segments.     

Physical linkage of variable, diversity and joining gene segments in the immunoglobulin heavy chain locus of the mouse

The variable region of antibody heavy chains is encoded by multiple variable (V), diversity (D) and joining (J) gene segments which seem to be assembled in a programmed developmental pathway during the maturation of B lymphocytes. A physical map linking V, D and J gene segments in the a haplotype of the murine Igh locus is presented in this report. The V gene located next to the known D and J gene segments maps 80 kb upstream of the JH locus and is a member of the VGAM3.8 V gene family. There is evidence for as yet unknown D gene segments located 5' to this V gene.     

Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality

Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)-based method to analyze T-cell receptor (TCR) gamma gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis.     

Somatic hypermutation of immunoglobulin variable region genes: focus on follicular lymphoma and multiple myeloma

Summary: Analysis of the rearranged immunoglobulin variable region gene hypermutation has provided important information concerning the clonal history and ontogenetic origin of various B-cell lymphoproliferative disorders. Under the selective pressure of antigen, mutational events in immunoglobulin genes will fine tune survival of B-cell clones bearing immunoglobulin with high affinity for antigen. Our studies aimed at analyzing neoplastic disorders originating from germinal and post-germinal center B-cells: follicular lymphoma and multiple myeloma. respectively. Despite the already acknowledged evidence for a selectable distribution of mutations within the clonal immunoglobulin variable heavy chain genes, very little is known about the contribution of light chains in the process of antigen selection. In follicular lymphoma. a more limited pattern of somatic mutation with less evidence of antigen selection was observed in variable K light chain genes (40%) than in their partner heavy chain genes (80%). In myeloma, hypermutation of variable light chain genes, with a distribution suggestive of antigen selection, was frequently observed. Based on these data and recent reports it appears that the light chain expressed by the clonogenic myeloma B-cells plays a pivotal role in the antigen selection process. Additionally, abortive K light chain variable region genes in X-expressing myeloma carried a significant number of somatic mutations indicating that the cell of origin is open to the hypermutation machinery at that particular developmental stage irrespective of antigen selection.     

Immunoglobulin VH genes in thymic MALT lymphoma are biased toward a restricted repertoire and are frequently unmutated

Thymic MALT lymphoma shows certain distinctive features among MALT lymphomas, such as expression of IgA isotype, consistent lack of API2-MALT1 gene fusion, and very strong association with autoimmune disease, especially Sjogren's syndrome. To help clarify the nature of the clonal lymphoid infiltrates, we analysed the usage and somatic hypermutation of the Ig heavy chain variable region (V(H)) genes in nine different cases. The V(H) rearrangement was potentially functional in all cases and was restricted to the V(H)3 family. V(H) usage was biased toward V(H)3-30 (five cases) and V(H)3-23 (three cases) segments, which have both been frequently expressed by autoimmune B cells. Somatic hypermutation was absent in five cases. Fewer than the expected replacement mutations were found in the framework regions in two cases, indicating a negative antigen selection pressure. Ongoing mutation was absent in all cases. D segment usage was varied, whereas J(H) segment usage was restricted to J(H)4. The observed patterns of V(H) usage and mutations suggested that specific antigens may play a pathologically relevant role in the genesis or progression of thymic MALT lymphoma.     

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

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