Pairing of VH gene families with the λ light chain: Evidence for a non-stochastic association

The frequencies at which four V_H gene families pair with the λ1 light (L) chain were determined by sequential hybridization of V_H- and λ1-specific DNA probes to mitogen-induced colonies of B cells. Analysis of pair frequencies indicates that the repertoire of λ L chain antibodies is generated by the stochastic pairing of smaller 3′-to-mid-locusV_H gene families (X-24, S107, Q52). However, the large 5′ V_H J558 family appeared to associate with the λ1L chain non-stochastically; the frequency of VhJ558/λ1⁺ colonies among all λ1⁺ colonies was significantly lower than the frequency of J558 expression among all (Cμ⁺) B cell colonies. This difference suggests that selection, either intrinsic at the level of rearrangement or heavy and L chain pairing, or extrinsic following surface immunoglobulin expression, may operate to shape the λ antibody repertoire prior to the introduction of exogenous antigen.     

Human Heavy Chain Variable Region Gene Diversity,Organization, and Expression

Elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire remains a major challenge in immunology. Knowledge of V gene diversity, organization, and expression is important to an understanding of the formation of the antibody repertoire in normal as well as diseased states. In the last few years, great advances have been made in our understanding of the human heavy chain variable region (V_H) gene locus. In this review we present the current knowledge of V_H gene diversity, organization, and utilization in normal individuals followed by a discussion of the possible relevance of these findings to autoimmunity.     

Comparative study of IgA VH3 gene usage in healthy TST− and TST+ population exposed to tuberculosis: deep sequencing analysis

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST⁺) and uninfected TST⁻) with M. tuberculosis. From a total of 22 V genes analysed, the TST⁻ population preferred the V_H3-23 and Vκ1 genes. The V_H3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST⁻ population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST⁺ population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST⁻ population. The antibodyome difference between both populations suggests a preference for antibodies with V_H3-23, D3-3, J_H4 gene usage by the TST⁻ population that could be associated with resistance to infection with M. tuberculosis.     

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.     

Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA)

The analysis of rearranged antibody-encoding genes from B cell foci in rheumatoid synovial tissue has characterized these cells as highly mutated memory B cells with a high proportion of members of the V_H4 family. In order to characterize further the V_H4 response in one patient, B cell-rich areas from different sections of synovial membrane (SM) were identified by CD20 staining, isolated by microdissection and pooled, in order to analyse highly enriched B cells without selection by in vitro culture procedures. From DNA of about 5 × 10³ B cells rearranged V_H genes were amplified by polymerase chain reaction (PCR) and cloned. Sequencing of 11 clones containing rearranged V_H4 gene products revealed that seven were potentially functional, and all were mutated with 84–96% homology to known germ-line (gl) genes and V_H4 gl genes amplified from the patient’s genomic DNA. Analysis of the complementarity determining region (CDR) 3 revealed that two products represented members of one B cell clone which differed by five nucleotide changes. Three of the five mutations encoded amino acid replacements in CDRs indicating antigen-driven expansion of one specific clone. Additional analyses of 25 members of three B cell clones from isolated aggregates showing intraclonal diversity in one of three clones provided further evidence that antigen selection takes place in the SM. Overall, the pattern of mutations and the replacement to silent (R:S) ratios were diverse, with six products indicating antigen selection by their high R:S ratios in CDRs. Although DNA analysis does not allow a characterization of antibody specificities, we can conclude from our analysis of antibody-encoding genes that selection by antigen and expansion of specific clones occur in the SM against the background of polyclonal activation.     

The Human VH Repertoire: A Restricted Set of VH Genes May be the Target of Immune Regulation

During the development of the immune system, a restricted set of V_H gene segments provides the bulk of the immunoglobulin heavy chain repertoire. Most of these V_H genes have been found later in life encoding autospecificities either in normals or in patients with autoimmune diseases. Additionally, there is considerable evidence that the fetal/neonatal B-cell repertoire is autoreactive and idiotypically connected. In the course of sequencing the heavy chain of a panel of human autoantibodies mainly derived from patients with autoimmune diseases, we found that one of the V_H families, and more specifically one single V_H gene contributes to a large extent to the adult autoimmune repertoire in restricted as well as unrestricted responses. This V_H gene segment is not particularly overexpressed in the fetus. Since the only common element to these autoreactive responses is the region encoded by the V_H gene itself, these observations may provide an important insight into B-cell regulation.     

Tracking of the V4–34 (VH4–21) gene in human tonsil reveals clonal isotype switch events and a highly variable degree of somatic hypermutation

The V_4–34 (V_H4–21) gene has been found to encode certain IgM autoantibodies, and is mandatory for pathological IgM anti-erythrocyte antibodies of I/i specificity. The gene is also commonly used by normal IgM-positive B lymphocytes, but its involvement in B cells which have undergone class switching to IgG or IgA is less clear. In order to track V_4–34 gene usage and class switching events during a normal immune response, we have probed RNA in a limited area of human tonsil. Results indicate that the V_4–34 gene undergoes class switching to IgG or IgA, with the progeny either remaining unmutated or containing large numbers of somatic mutations. Mutational patterns indicate possible ‘hot spots’, and some mutations appear deleterious. At the level of individual B cells, we have tracked a clonal isotype switch event from IgM to IgA, with each retaining close to germ-line configuration. In addition, we have followed a clonal switch from a mutated IgM to IgG, with no further accumulation of somatic mutations. These data indicate that the V_4–34 gene is involved in a maturing immune response, and that the routes to production of IgG or IgA antibodies are various.     

VH gene-family representation in peripheral activated B cells from systemic lupus erythematosus (SLE) patients

A semiquantitative polymerase chain reaction (PCR) assay described in this study has been used to analyse the V_H1, V_H3 and V_H4 repertoire expressed by total IgM⁺ and IgG⁺ B cells from normal individuals and lupus patients. This approach consists of a combination of B cell selection, utilization of the anchored PCR technique to avoid technical bias in the amplification of different V_H gene family cDNA templates, and screening of the amplified IgM or IgG cDNA rearrangements by family-specific oligonucleotide probes. In four lupus patients, V_H family representation in IgM⁺ and IgG⁺ in vivo activated B cells, selected by anti-CD71 antibody, and in total CD19⁺ B cells were compared. In all patients, V_H4 gene family segments were preferentially underrepresented in IgM⁺ activated B cells. In IgG⁺ B cells the results suggest that V_H4 expression is variable, depending on the phase of the disease. Polyclonal B cell activation, which is usually considered as being the first event in autoantibody production in SLE, cannot explain our results. The data evoke the possible involvement of a V_H4-specific B cell superantigen in the onset or development of SLE. This hypothesis is also supported by the sequence conservation of the fourth β loop—a putative superantigen binding site—of functional V_H4 gene segments which are preferentially used by anti-dsDNA lupus antibodies of established clones and hybridomas.     

Analysis of VH genes rearranged by individual B cells in dermal infiltrates of patients with mycosis fungoides

In patients with cutaneous T cell lymphomas such as mycosis fungoides B cells can frequently be detected in the lymphocytic dermal infiltrate. To analyse their immunoglobulin heavy chain gene repertoire, single B cells were obtained from tissue sections of two typical patients with mycosis fungoides using hydraulic micromanipulation followed by specific amplification of the respective gene segments by single-cell polymerase chain reaction (PCR) technique. A total of 21 V_HDJ_H genes was sequenced. From each individual B cell a single productive V_HDJ_H rearrangement was obtained. There was no clonal relationship detected between any of these rearrangements suggesting polyclonality of the infiltrating B cells. The representation of V_H families was in accordance with the germ-line complexity. A remarkably high number of V_H genes (5/13 in patient 1; 3/8 in patient 2) was completely or nearly germ-line-identical. Five of seven V_H4 family genes were nearly unmutated. On the other hand, most of the V_H3 gene family members were somatically mutated in an antigen-driven manner. The proportion of germ-line-identical V_H genes, the usage of individual V_H, D, J_H gene elements, and the pattern of somatic mutations found in the B cells infiltrating skin lesions of patients with mycosis fungoides resembles the peripheral blood repertoire, suggesting a bystander role of these cells.     

Cytoplasmic expression and specific binding of the VH/VL single domain intrabodies in transfected NIH3T3 cells

Intracellularly expressed antibody fragments (intrabodies) have been utilized as powerful tools not only for clinical applications but also for the functional analysis of proteins inside the cell. Among several types of intrabodies developed so far, single domain types composed of only the variable regions (V_H or V_L) of antibodies are the smallest and thus the easiest to design. In this study, four types of single domain intrabodies were evaluated against a cytosolic protein, Wiskott-Aldrich syndrome protein (WASP), in gene-transfected NIH3T3 cells. These single domains were composed of the V_H and V_L region with or without their leader sequences. Although these single domains were expressed at similar levels in NIH3T3 cells, the binding activity to the cytosolic target was higher in the single domain constructs with leader sequences. These results suggest the usefulness of the single domain intrabody constructs to analyze the functional domains of cytosolic proteins in cells.     

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.     

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V_HDJ_H, VκJκ and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V_HDJ_H, VκJκ, and c-myc gene sequences. Tumor-site specific V_HDJ_H, VκJκ, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V_HDJ_H, VκJκ, and c-myc mutations displayed predominance of transitions over transversions. The “neoplastic” V_HDJ_H sequence was expressed by about 10⁻⁵ cells in the bone marrow, and contained two of the three orbital, but none of the testicular V_HDJ_H mutations. The nature and distribution of the Ig V(D)J mutations found in the κ chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V_HDJ_H, VκJκ, and c-myc genes with comparable efficiency and modalities.     

Map position and usage of 3' VH family members: usage is not position dependent

The primary repertoire in mice, in large part, is determinedby the Ig gene segments joined to form the variable region genes—VDJfor the heavy chain genes and VJ for the light chain genes.However, the mechanisms that determine which V_H gene, of severalhundred available, is joined to a DJ_H structure remain unexplained.One theory proposes that the V_H gene segments closest to the3' end of the V_H locus are chosen because of their location,i.e. that proximity to a DJ_H structure is a prime determinantfor V_H selection. Alternatively, the content of the DNA maybe the determining factor regardless of chromosomal position.According to this hypothesis 3' preference is due to the coincidenceof preferred content and proximity to DJH structures. Sincethe 3' V_H families span more than a centimorgan of DNA we wereable to distinguish between these concepts by determining V_Husage of gene segments within this region. The BALB/c strainis ideal for this study since it uses 3' V_H families at a veryhigh frequency. We therefore mapped the positions of the 3'V_H gene families and used this map to investigate the usageof these gene segments in BALB/c fetal liver derived A-MuLVtransformed cell lines. We found that while V_H gene segmentsthroughout the Ig locus, both 3' and 5' were used, there wasa clear preference for 3' V_H families. However, within the 3'V_H segments the most proximal were not preferred, indicatingthat proximity to DJ_H structure was not a strict determinantof V_H utilization.     

Selection of VH gene repertoires: differentiating B cells of adult bone marrow mimic fetal development

In the present study we have compared by in situ hybridizationand by a CFU-B colony assay V_H family usage in the pre-B andB cell compartments of the bone marrow of adult BALB/c and C57BL/6mice. We have found that the position dependent increased expressionof the V_H 7183 family, observed in neonatal mice, is characteristicof early differentiating B cells of adult mice. The quantitativeanalysis for each V_H family of the number of pre-B and B cellsproduced daily and the number of mature B cells present in theperipheral immunocompetent cell pool of adult BALB/c demonstratesthe existence of selecting mechanisms operating within the bonemarrow at the level of the emergent repertoire and at the levelof export of newly formed cells into the periphery. This selectiveprocess results in the decreased peripheral representation ofthe V_H 7183 family and in the accumulation of cells belongingto the other V_H families. Selection of V_H family usage in peripheralrepertoires may be determined according to lymphocyte life-span,as we have found a preferential utilization of the V_H J558 familyin populations of partially enriched, long-lived B cells.     

A Mouse with a Monoclonal Primary lmmunoglobulin Repertoire not Further Diversified by V-Gene Replacement

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a λ1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a “knocked-in” V_HDJ_H rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB''s primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 λ light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.     

Rearrangement of upstream DH and VH genes to a rearranged immunoglobulin variable region gene inserted into the DQ52-JH region of the immunoglobulin heavy chain locus

We investigated gene rearrangements in the mutant IgH locus of a mouse strain generated by insertion of a rearranged heavy chain variable region gene (V_T15) into the DQ52-J_H region through gene targeting. In more than half of the B cells of heterozygous mutant mice, the mutant IgH locus was silenced by the rearrangement of an endogenous D_H or D_H and V_H gene to the inserted V_T15 gene. In these cases, a functional V_HD_HJ_H gene was present on the wild-type allele. The silencing rearrangement appeared to be mediated by recombination signal sequence (RSS)-like elements present in the “recipient” V_T15 gene. Among the many such elements on the inserted V_T15 gene, which apparently met the requirement for an RSS with respect to nucleotide sequence, only two were observed in the actual rearrangements. This indicates that targeting of the recombination machinery involves sequences in addition to the RSS motifs as they have been characterized so far. In homozygous mutant mice, most B cells appeared to carry the intact V_T15 gene on both mutant IgH alleles, although single-cell polymerase chain reaction revealed that silencing rearrangements occured frequently in B cell progenitors in the bone marrow. This observation indicates that once silencing rearrangements are initiated in a cell, they involve both V_T15 genes in most cases, reminiscent of normal D_H-J_H rearrangement. B cells which did not initiate such rearrangements develop to populate the peripheral B cell compartment.     

Strong association of HLA-B27 heavy chain with β2-microglobulin

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with β₂-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with β₂-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/β₂-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.     

Selective activation of VH3A10+ rheumatoid factor producing B cells by staphylococcal enterotoxin D

Staphylococcal enterotoxin D (SED) is a T cell superantigenwhich selectively targets ß TCRs bearing particularV^ß elements. A second function of SED relates to thepreferential activation of a B cell subset characterized bya high frequency of rheumatoid factor (RF) producing B cells.To define the molecular basis of the SED-induced B cell repertoireshift, we have analyzed Ig heavy chain genes in B cell clonesexpanded after SED stimulation and compared them with B cellclones established in the presence of anti-CD3 stimulated helpercells. Gene segments of the V_H3 family were most frequentlyutilized under both stimulation conditions (42% anti-CD3; 47%SED). Sequence analysis of V_H3 gene segments demonstrated thatthe repertoire of V_H3 elements in B cell clones from SED drivenand anti-CD3 driven cultures were distinct (P=0.01). RF activitywas closely associated with the expression of selected V_H3 elements.B cell clones stimulated with SED preferentially expressed V_H3A10,whereas V_H26 was the gene segment dominantly used in B cellclones expanded with anti-CD3 stimulated helper cells. The usageof J_H and D_H elements was indistinguishable in SED and anti-CD3driven B cell clones, suggesting that SED targets V_H3⁺ B cellsthrough a V_H-specific mechanism. Comparison of the closely relatedsequences of the SED responsive V_H3A10 and the SED non-responsiveV_H26 element suggested a role of a sequence polymorphism inthe CDR2 reminiscent of B cell reactivity to conventional antigens.In contrast to conventional antigens, SED can induce differentiationof a high frequency of naive B cells. Thus, this staphylococcalenterotoxin combines selective activation of T cells with selectiveactivation of B cells and might be able to direct T cell helpto RF producing B cells.