Three New Cross-Reacting Idiotopes as Markers for the Products of Two Distinct Human VH3 Genes Expressed in the Early Repertoire

This article describes characterization of three new cross-reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing V_H3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) V_H3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti-idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fcα, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti-idiotypic MoAbs were shown to be V_H-specific reagents by the independence of their reactivity upon the L-chain type, or the untigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for V_H3 gene-products was demonstrated by its sole cross-reactivity with V_H3 proteins. Each anti-Id had a different reactivity pattern with a panel of MoAbs utilizing different V_H3 genes. By relating the V_H sequences of the tested V_H3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47-encoded proteins, although 3H1 had weak cross-reactivities with a few other V_H3 gene-products. 7B4 appeared to be specific for antibodies utilizing DP46-related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize V_H3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46-related gene products, B6 and D12 additionally recognized non DP46-related proteins and were thus different to 7B4.     

A Human Rheumatoid Factor C304 Shares VH and VL Gene Usage with Antibodies Specific for Ubiquitous Human Viral Pathogens

Analysis of the variable region getie sequetices of a human hybridoma rheumatoid factor (RF), derived from a patient with rheumatoid arthritis (RA), revealed the expression of genes from the V_βI and V_H3 families. Specifically, the C304 RF had rearranged the DPL8/Humlv1042 and VH26 germline V_L and V_H genes, respectively. This gene usage has been observed in the rearrangement of human anti-viral antibodies specific for the herpes group of viruses. This overlap between the autoimmune and anti-viral antibody gene repertoires suggests a possible structural relationship between the immune response directed against ubiquitous pathogens and the induction of RF production.     

Predominant V-Region Gene Configurations in the Human Antibody Response to Haemophilus influenzae Capsule Polysaccharide

The antibody response to Haemophilus influenzae type b polysaccharide (Hib PS) is known to be encoded by a few V-region genes. We have obtained four human monoclonal Hib PS antibodies from four healthy adult subjects immunized with diphtheria toxin-conjugated Hib PS vaccine. The V_H gene segments that encode for these antibodies belong to the V_H3 gene family, of which two are related to the V3-23 gene and two to the V_H3b subfamily. Both hybridomas that express a V3-23-related gene use short D-segments (3 bp), the J_H6 gene segment and a V_k gene derived from the A2 germline gene. The two hybridomas that express V_H3b genes use D-segments of conventional length (24–33 bp), the J_H4 gene segment and a non-A2 V_k gene. Comparison of our sequences with those reported by others suggests that the above patterns of V-region gene segment association exemplify two V-region gene configurations that are predominant in the Hib PS antibody response. The first configuration is reminiscent of antibodies produced by B-l B cells while the second is more characteristic of antibodies produced by conventional B cells. The possibility that these two configurations, in fact, represent the products of two different B cell lineages remains to be elucidated.     

Selection during development of VH11+ B cells: a model for natural autoantibody-producing CD5+ B cells

Summary:  Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5⁺ B-cell-derived natural autoantibody, the V_H11V_κ9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5–10% of the CD5⁺ B-cell subset, with a large portion accounted for by two novel BCRs, V_H11V_κ9 and V_H12V_κ4. We have found that the development of B-lineage cells with a V_H11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i) one of the most common V_H11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii) cells containing V_H11-µ chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow V_H11 rearrangements; and (iii) only a subset of V_κ-light chains combine with V_H11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain V_H11 generation to fetal development and may favor production of B cells with the prototype V_H11V_κ9 BCR.     

Positive selection focuses the VH12 B-cell repertoire towards a single B1 specificity with survival function

Summary:  B cells of varying antigen specificities are consistently present in the unmanipulated repertoire. These B cells appear to belong to the marginal zone (MZ) and B1 B-cell subsets and provide protection to the blood and lymph, respectively. Some are specific for self-antigens, suggesting that they are selected based on specificity for self but have a protective role against foreign pathogens. One of these specificities is for phosphatidylcholine (PtC). Anti-PtC B cells comprise 5–8% of the B1 repertoire and are protective against bacterial pathogens. In general, they are restricted to the expression of two V_H/Vκ combinations, V_H11/Vκ9 and V_H12/Vκ4/5H. This review focuses on the differentiation of V_H12 anti-PtC B cells. They undergo a series of positive selection events beginning at the pre-B-cell stage that enriches for those with a V_HCDR3 and L chain required for PtC binding and eliminating the majority of V_H12 B cells that lack the ability to bind PtC. Thus, positive selection focuses the V_H12 repertoire toward PtC, ensuring that anti-PtC V_H12 B cells are a significant component of the B1-cell repertoire in all individuals.     

Somatic Hypermutations in the Immunoglobulin Genes of Two New Human Lymphoma Lines of Lymphatic Follicle Origin

Variable immunoglobulin heavy-chain regions (VDJ) of two newly established human lymphoma cell lines (HF-1 and HF-4) were sequenced. The most homologous germline V_H gene found for both the HF-1 and HF-4 sequences was V_H26 of the V_H3a (V gene) family (82% and 91 % homologies, respectively). The J_H region of the HF-4 heavy-chain sequence contained two nucleotide differences compared to the published germline J_H3 gene. The D_HJ_H region of the HF-1 gene had a record high number (20%) of somatic mutations.
The numerous hypermutations found in the HF-1 cell line support the hypothesis that in some human follicular lymphomas, mutations continue to accumulate in immunoglobulin genes during the malignant growth. Follicular lymphoma cell lines, which have an active mutational machinery, in future may help to solve the molecular events behind the somatic hypermutations modifying immunoglobulin genes of B lymphocytes.     

VH-Gene Family Dominance in Ageing Mice

The cellular composition and Vn-gene family repertoire were compared in different B-cell compartments from young adult (8–12 weeks) and old (18–24 months) C57BL/6 and BALB/c mice. Ageing mice were found to have a higher frequency of peripheral mature B cells utilizing genes from a single V_H-gene family. While in each individual old C57BL/6 mice cells expressing the V_H J558 gene family consistently were over-represented, a marked individual variation was observed in old BALB/c mice with increased frequency of either the V_h J558, Q52 or J606 families. Aged mice were found also to have a reduced number of bone-marrow pre-B cells and an augmented number of splenic Ig-secreting cells. These results suggest that old mice express less diversified antibody repertoires possibly as a consequence of reduced input from precursors and increased peripheral selection, which may be responsible for the progressive establishment of immunodeficiency.     

Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human V rearrangements

Summary: B cells are unique in that they generate and tolerate a high rate of mutations in their antigen receptor genes and employ these mutations as a basis of avidity maturation. The precise role of the mutational machinery versus subsequent selection in determining the frequency and distribution of mutations has not been fully analyzed. To address these issues, the influence of the intrinsic mutational machinery and subsequent selection on the frequency and distribution of mutations in the expressed human immunoglobulin repertoire was analyzed. Analysis of non-productively rearranged v_H genes from individual human B cells provided an opportunity to examine the immediate impact of somatic hypermtitation without superimposed selective influences. Comparison with the frequency and distribution of mutations in the productively rearranged human V_H genes permitted an estimate of the influences of subsequent selection.     

Identical VHD and DJH Junctions in Monoclonal Antibodies Derived in Response to Dextran B512 Could be the Result of Developmental Selection

We describe here the CDR3s of a collection of monoclonal antibodies (MoAb) with specificity for the carbohydrate dextran B512 produced in the mouse strain C57BL/6. In spite of the postulated mechanisms for variability in this region, a high proportion of these monoclonals displayed identical V_HD (24/30) and DJ_H (21/30) junctions and 21 of them were identical in the whole CDR3. These 21 independently generated identical CDR3s could be ordered in eight groups indicating that not a particular CDR3, but instead the mechanism for generating identical junctions was preserved. Two of the CDR3s in this study were found to be identical to the CDR3 of the monoclonal B1-8 produced in C57BL/6 in response to proteins bearing the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). This and other parameters support the notion that the generation of identical junctions could be independent of antigenic selection. We also report here the association between J_H usage and amino acid (aa) residues at the V_HD and DJ_H junctions. Since these MoAb were generated in response to dextran B512, immunoglobulin conformation has to be compatible with antigen binding. Nevertheless, no aa residue of CDR3 could be directly related to antigen binding. We postulate therefore, that the observed selection of CDR3s could be directed to the production of variable regions with protein configuration most suitable with immunoglobulin folding and may occur prior to antigenic selection. Selection for junctional residues in relation to J_H usage and the generation of identical CDR3s are probably different events. Possible genetic mechanisms operating for CDR3 construction and/or selection by cellular ligands are discussed.     

The Cross-Reactive Idiotopes Recognized by the Monoclonal Antibodies 9G4 and LCI are Located in Framework Region 1 of Two Non-Overlapping Subsets of Human VH4 Family Encoded Antibodies

The monoclonal anti-idiotopic antibodies LCI and 9G4 bind two non-overlapping sets of V_H4 encoded antibodies. 9G4 exclusively binds V_H4–21 encoded antibodies, while LCI binds antibodies derived from V_H4 family gene segments V71-2, V71-4, V_H4-18, V_H72-I and V2-1. The V_H4–21 gene segment is utilized by most cold agglutinin (CA) antibodies with I/i specificity, while antibodies encoded by other V_H4 gene segments are associated not with CA disease, but primarily with rheumatoid-factor (RF) activity.
We previously determined that the idiotope to which 9G4 binds in V_H4-21-derived antibodies is located in framework region l (FR1). In the present study, by using mutational analysis involving individual framework- and complementarity-determining region exchanges between V_H4-2I- and V71-2-encoded antibodies, we have found that the idiotope to which LCI binds in V71-2-derived antibodies also maps to FR1. The LC1 idiotope is heavy (H)-chain associated, but requires pairing with a light (L) chain for LCI binding. Recombinant antibodies composed of a variety of kappa (k) and lambda L chains paired with either a V71-2 or V_H4–21 chain were produced in the baculovirus expression system. LCI bound all of the k-containing antibodies but did not bind the V71-2-encoded H chain alone nor to the two A-containing antibodies. This experiment demonstrates that not all light chains exert equivalent influence on the conformation of the H-chain idiotope. These results indicate that the FR1 of V_H4-encoded antibodies is immunogenic and suggest a physiological role of FR l during an immune response.     

Monozygotic Rheumatoid Arthritis Twin Pairs Express Similar Levels of Conserved Immunoglobulin V Gene in Polyclonal Rheumatoid Factors Irrespective of Disease Status

The degree of polyclonal RF heterogeneity was assessed in diseased and non-diseased twins with rheumatoid arthritis (RA). The distribution of variable region determinants encoded by a set of immunoglobulin germline, or minimally mutated germline, genes within IgM RF, IgG RF and IgA RF isotypes was determined by ELISA using specific mouse monoclonal antibodies (MoAb) in fractionated plasma from 12 members of six monozygotic twin pairs with RA, The results reveal that at least 40% (range ∼ 18–87%) of IgM RF are encoded by a small set of ∼ 10 genes from the V_H1, 3 and 4 families. Furthermore, a significant proportion of IgG RF and IgA RF (∼ 30%) are also encoded by these same genes. Comparison with RF-negative fractions of immunoglobulins showed that the examined variable region determinants were overrepresented in the RF fractions.
The level of expression of the variable region determinants in RF were generally similar within twins but different between unrelated twin pairs irrespective of disease status. The variability of V_H gene usage between unrelated individuals suggests that the level of expression and regulation of the variable region determinants may be genetically regulated or influenced by common environmental factors.     

Distribution of Heavy-Chain Variable-Region (VH) Subgroups on Human Lymphocytes

Lymphocytes from 20 normal blood donors were stained with fluorescein isothiocyanate (FITC)-conjugated anti-F(ab')₂, anti-V_HI, anti-V_HII, and anti-V_HIII subgroup antisera. The means of the percentages of staining were 2.9%, 5.0%, and 5.5% for the V_HI, V_HII, and V_HIII subgroups, respectively. The sum of the percentages of the lymphocytes stained with each of the V_H subgroup-specific antisera corresponded well with the percentages of lymphocytes stained with an anti-F(ab')₂ antiserum. In addition, it was shown by double immunofluorescence staining and in various depletion experiments and tests with thymocytes and lymphocytes from patients with hypogammaglobulinemia that the cells staining with anti-V_H antisera corresponded to the membrane Ig-positive cells—that is, the B-lymphocyte population.     

Clonotypic Dominance and Variable Gene Elements of Pathogenic Anti-DNA Autoantibodies from a Single Patient with Lupus

This study explores the usage and diversity of the variable gene elements expressed by human lupus antibodies to DNA bearing the 0–81 idiotype, a marker of pathogenic anti-DNA autoantibodies. Rather than studying DNA-specific clonotypes from different patients, a panel of idiotype positive anti-DNA autoantibody-secreting clones from a single individual were analysed. By cloning and nucleotide-sequeneing the heavy-chain variable gene segments, evidence was found for dominance of clonotypic patterns. Also noted was a high rate of diversification among the variable (V_H), diversity (D_h) and junctional (J_H) gene segments utilized, with a pattern of mutations indicative of antigenic selection. These features suggest that the clones secreting the lupus pathogenic autoantibodies have been selected over multiple generations through an affinity-maturation process that is reminiscent of antigen-driven immune responses.     

Nucleotide Sequence Analysis of Rheumatoid Factors and Polyreactive Antibodies derived from Patients with Rheumatoid Arthritis Reveals Diverse Use of VH and VL Gene Segments and Extensive Variability in CDR-3

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of V_H and V_L gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of V_H3 genes beyond that one would expect based on random utilization.     

Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes

Summary: Recombination of V_H D_H and J_H genes is a unique first step in normal B-cell development. Subsequent differentiation to a mature plasma cell is accompanied by further events in the Ig genes, including V_I-J_t joining, somatic hypermutation and isotype switching. Chromosomal changes leading to B-cell tumors can occur at many points in this sequence, and may be partly a consequence of the genetic mobility and mutability permitted in order to generate a diverse antibody repertoire, V genes of neo-plastic B cells may reflect the point of maturation reached by the B cell of origin, prior to transformation, Analysis of tumors therefore provides useful information on V-gene patterns in normal B cells, and may add another dimension to classification of B-cell tumors. Transformation ma)' also preserve cell populations normally destined to die by apoptosis. Tumor cells arrested in the sire where somatic hypermutation and isotype switch are occurring can still be subject to these processes, and could be influenced by persisting antigen. However, mutation is silenced at the point of exit lo the periphery, leading lo fixed mutational patterns in tumors of mature B cells, V-gene analysis provides an invaluable tool for understanding the genesis of neoplastic change. It also has a clear clinical relevance in tracking tumor cells, measuring residual disease, and finally in offering the opportunity of developing vaccines for treatment.     

Molecular Characterization of Immunoglobulin Variable Regio from Murine Monoclonal Antibodies Specific for Simian Virus 40 Large Tumour Antigen

In this report we compare the immunoglobulin variable (IgV) region function and structure relationships of murine monoclonal antibodies that recognize simian virus 40 (SV40) large tumour antigen (T ag). Comparison of monoclonal antibody V region function is based on SV40 T ag epitope recognition and idiotype (Id) expression. Structural comparisons are based on V region gene sequence determination. The data presented herein, suggests that a high degree of homology within both the V_k and V_H regions, along with minor differences within V_k complementarity determining regions (CDR) may result in the detection of similar SV40 T ag epitopes by the monoclonal anti-SV40 T ag preparations. The expression of a cross-reactive Id also appears to be based on the high degree of homology within both V_K and V_H regions and depends on conformational interactions imparted by both regions.     

Role of human FcεRI+ cells in HIV-1 infection

Summary: Enhanced serum IgE levels in adults and children with HIV-1 infection could be a marker of poor prognosis. HIV-1 infection is believed to involve a switch toward a "T_H2-like" cytokine pattern. HIV-1 gp120 from different clades is a potent stimulus for histamine release from human basophils and mast cells. Gp120 also induces IL-4 and IL-13 synthesis from basophils. It functions as a viral superantigen by interacting with the V_H3 region of IgE to induce mediator release from human FcεRI⁺ cells. The chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES, is expressed by basophils and lung mast cells. By interacting with the CCR3 receptor on FcεRI⁺ cells, HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells. Tat protein also induces IL-4 and IL-13 release from basophils. Incubation of basophils with Tat protein upregulates the surface expression of the CCR3 receptor, a co-receptor of HIV-1 infection. Extracellular Tat affects the directional migration of human FcεRI⁺ cells, CCR3 expression and T_H2 cytokines release. We have shown that HIV-1 proteins gp120 and Tat trigger the release of cytokines critical for T_H2 polarization from FcεRI⁺ cells through two distinct mechanisms. In addition, Tat upregulates the β-chemokine receptor CCR3, making FcεRI⁺ cells more susceptible to infection with CCR3 tropic HIV-1 isolates.
This paper is dedicated to Rita Levi-Montalcini who first suggested an involvement of FcεRI⁺ cells in HIV-1 infection. This work was supported by a grant from the Istituto Superiore Sanità (AIDS project 40B.64 and 40A.67), CNR (Target project Biotechnology No. 99.00216.PF31 and No. 99.00401. PF49) and MURST (Rome, Italy).     

The Structure of the Variable Regions of Mouse Monoclonal Antibodies to Hepatitis B Virus Core Antigen

From a panel of monoclonal antibodies (MoAbs) directed against E. coli -derived native and denatured hepatitis B virus (HBV) core antigen we have selected a set of specific MoAbs which recognize different linear antigenic determinants: MoAb C1-5—cl epitope; MoAb 14K8—less immunogenic N-terminal region; and MoAbs 13C9, 10F10 and 14E11, 14G3—the immunodominant region between amino acids 134 and 140.
We have applied the polymerase chain reaction technique to clone Ig V_H and Y_L, region genes, and appropriate full-length cDNA clones were obtained and characterized by nucleotide sequence analysis. Among the six heavy chain variable region sequences examined, three Y_h families were represented. Two of them belong to the 7183 (MoAb Cl-5) and 3609 (14B8) families respectively and four, having only two amino acid changes in the CDR2 region, to the J558 family. These four probably are derived from a single expanded B-cell clone. The light chain sequences indicate that their YL are encoded by Y_K21, V_K19 and Y_K3 germline genes. Unlike Y_H genes, light chain genes are closely related to known representatives of mouse kappa light chain families and are employed also by MoAbs raised against other antigens.