Variable Region Gene Usage Research Articles

Restricted variable region gene usage and possible rheumatoid factor relationship among human monoclonal antibodies specific for the AD-1 epitope on cytomegalovirus glycoprotein B

The nucleotide sequences of the variable region genes encoding five different human, high affinity antibodies, specific for the major neutralization determinant (AD-1) expressed by human cytomegalovirus glycoprotein B (gp58/116), have been determined. Three of the five heavy chain variable regions belonged to the small V HV-family, although they combined with a diverse set of light chains (V κIIIb, V λII and V λIII). The other two antibodies belonged to V H-families III and IV. One of the V HV-family genes most likely originated from a previously unreported germline gene or allele, since it carries a nine nucleotide insert in framework 1. In addition, V λ-genes showed variable homology (77–95%) to known germline sequences, while V κ-genes showed high homology (approximately 98%) with their proposed germline origin. Despite the close homology of the V kIIIb-gene used to express one of the antibodies with its corresponding germline gene, the protein did not strongly express some idiotypes associated with this light chain family. There is, thus, no direct relation between the expression of these crossreactive idiotypes and the use of even modestly mutated light chains belonging to this V κ-family, which has been implicated in the development of anti-idiotypic networks possibly inducing autoantibodies, such as rheumatoid factors.

Variable region gene usage of human monoclonal rheumatoid factors derived from healthy donors following immunization

A study was performed to compare the use of immunoglobulin V gene segments by rheumatoid factors (RF) produced in physiological responses following a defined antigenic stimulus, with RF produced in rheumatoid arthritis (RA) and RF produced as monoclonal (M)-components in certain lympho-proliferative diseases. A panel of 46 monoclonal RF was produced, using hybridoma techniques, from healthy individuals following immunization with foreign antigens (mis-matched red blood cells). A panel of previously characterized monoclonal RF from RA synovial tissues was extended to a total of 24 and included in the study. The variable heavy (VH) and variable light (VL) chain gene families used by these RF were determined using idiotypic markers and polymerase chain reaction amplification with VH-specific primers. The frequencies of expression of the various gene families was compared between the two groups, and compared with the published expression frequencies seen amongst M-component RF. The majority (87%) of RF from healthy donors were found with light chains using V gene segments of the V chi 3 family, in conjunction with VH gene segments belonging to the VH1, VH3 and VH4 families. The over-expression of V chi 3, together with the distribution of VH families, demonstrates close similarities with RF found as M-components in lympho-proliferative diseases. In contrast, RF from RA patients showed a predominant use of VH3 gene segments (82%) and an unbiased expression of V chi 3 segments (29% of the chi light chains). These data suggest that RF found as M-components are representative of RF used in normal physiological responses, but have undergone neoplastic or other transformation. RF found in the synovial tissue of RA patients appear to be driven by different mechanisms than RF seen in physiological responses in healthy individuals.

T-cell receptor variable region gene usage by CD4+ and CD8+ T cells in bronchoalveolar lavage fluid and peripheral blood of sarcoidosis patients.

Sarcoidosis is a chronic noncaseating granulomatous disease of unknown etiology. An accumulation of CD4+ T cells in the alveolar space of the lungs is a characteristic feature of the disease. We have in this study analyzed T-cell receptor (TCR) variable region (V) gene usage by CD4+ and CD8+ lung and peripheral blood T cells of 29 sarcoidosis patients and 15 control subjects. In the patient group, we found a 100% positive correlation between TCR V alpha 2.3+ CD4+ lung T-cell expansions and the expression of the HLA-DR3(17),DQ2 haplotype. The remaining TCR V alpha/V beta gene products analyzed in this study--V alpha 12, V beta 2, V beta 3, V beta 5.1, V beta 5.2/5.3, V beta 5.3, V beta 6.7, V beta 8.1, and V beta 12--were in general normally expressed by CD4+ T cells, although some of them were used to a significantly higher or lower degree by lung T cells compared to peripheral blood T cells. We also performed repeated TCR V gene analyses on some HLA-DR3+ patients and found an association between the ratio bronchoalveolar lavage fluid/peripheral blood V alpha 2.3+ CD4+ T cells and clinical signs of disease activity. Finally, when analyzing TCR V gene usage by CD8+ bronchoalveolar lavage fluid and peripheral blood T cells, a normal V alpha 2.3 usage was found in all cases, but lung-restricted T-cell expansions using other TCR V gene segment products were identified.

Heavy and light chain variable region gene usage in antibodies specific for various human class I and class II HLA molecules

Heavy and light chain variable region gene usage in antibodies specific for various human class I and class II HLA molecules

Molecular characterization of human antibodies to bacterial antigens: Utilization of the less frequently expressed V H2 and V H6 heavy chain variable region gene families

Structural analysis of the human immunoglobulin repertoire holds promise for determining the basis of variable region gene usage in response to a variety of auto and exogenous antigens. Here we report the nucleotide sequences of the heavy and light chain variable regions expressed by three human monoclonal antibodies specific for two clinically relevant bacterial pathogens, Bordetella pertussis and Haemophilus influenzae type b. The cell lines were derived by in vitro stimulation of lymphocytes from spleen or tonsillar tissue, respectively, and bind to different antigens from the two organisms. The single B. pertussis antibody is of the IgMγ. isotype and utilizes the single V H6 gene segment in combination with a V γ2 gene and demonstrates limited somatic mutation, yet is highly indicative of an antigen-driven immune response. One H. influenzae antibody is of the IgG2γ isotype and expresses a V H 3 gene segment with a V γ 1 gene, while the second cell line produces an IgG3 γ antibody expressing a combination of V H2 V γ3 . Both molecules show evidence of somatic mutation. The D gene segments of the heavy chains vary in length and display limited sequence homology with known germline D segments. As demonstrated previously, J H4 predominates (two J H4 and one J H3) and all three utilize the J γ3 gene segment. In addition, we have isolated and sequenced a number of germline V H2 gene segments in an attempt to better understand the nature of the V H2 germline repertoire. In addition to contributing to the understanding of the human antibody repertoire, such clinically relevant molecules may prove to be a source of passive immunotherapy for those at risk to developing disease.

3 Normal B lymphocyte differentiation

Normal differentiation of B lineage cells has been the subject of intensive investigation over the past three decades. Current models of this process in humans are melded from the results of studies in a variety of organisms, including humans, mice and birds. Several recent developments have significantly reshaped and refined these models. The technique of homologous recombination in embryonic stem cells has allowed the production of mice with selectively disrupted genes that are important for B cell development in mice. At the same time, functional studies of human B cell differentiation, together with analysis of naturally occurring mutations that disrupt this process, have progressed rapidly. This has provided insight into the pathogenesis of lymphoproliferative and immunodeficiency diseases as well as a clearer view of normal developmental events. In this chapter we have reviewed human B cell differentiation with particular emphasis on newly emerging concepts. We also discussed CD5, a pan-T cell antigen that is expressed in low levels on a subpopulation of B cells implicated in the pathogenesis of chronic lymphocytic leukaemia (CLL). Finally, we discussed the issue of restricted variable region gene usage during B cell ontogeny and in CLL.

Biased T cell receptor V gene usage in rheumatoid arthritis. Oligoclonal expansion of T cells expressing V alpha 2 genes in synovial fluid but not in peripheral blood.

To analyze the T cell receptor (TCR) variable (V) region gene usage in the rheumatoid joint. Monoclonal antibodies (MAb) were used to determine the prevalence of selected V elements on T cells in synovial fluid (SF) from rheumatoid arthritis (RA) patients and in peripheral blood (PB) from RA patients and normal controls. V alpha 2-positive PB and SF T cells from 1 patient were cloned by immediate limiting-dilution and analyzed by restriction mapping. In 9 of 14 RA patients, SF was enriched in at least 1 of the selected V elements, compared with PB. TCR genes of the V alpha 2 family were the most frequently overrepresented in the SF (4 patients). The expanded V alpha 2-positive cells were oligoclonal in SF but heterogeneic in PB. Our data showing biased and clonally restricted TCR elements in the rheumatoid joint indicate major histocompatibility complex-restricted antigen recognition, rather than a "superantigen," in the pathogenesis of RA.

Diversity of Immunoglobulin Light Chain Usage in the Human Immune Response to Haemophilus influenzae Type b Capsular Polysaccharide

The response to the capsular polysaccharide of Haemophilus influenzae type b (Hib PS) has been used to determine the molecular basis of antibody gene diversity in humans. In contrast to the relatively restricted nature of anti-Hib PS heavy-chain variable region gene expression, a variety of light-chain variable region genes may encode this antibody (Ab) response. Light-chain variable region gene usage appears to determine the expression of certain Ab idiotypes and fine antigen specificity. To further define the role of light-chain variable region gene usage in important anti-Hib PS Ab subgroups, we have cloned and sequenced a number of immunoglobulin light-chain variable region genes (IgVL) from human monoclonal IgA anti-Hib PS Ab generated in response to Hib PS-protein conjugate vaccines. Three of these Ab are encoded by unusual variable segments. One kappa-Ab is encoded by the "predominant" V kappa II A2 germline gene but, in contrast to a previously reported A2-encoded IgVL sequence, differs from the A2 germline sequence. The IgVL sequence of a second Ab is the only sequence of a kappa-Ab that cross-reacts with the structurally related antigen Escherichia coli K100 polysaccharide reported to date. This IgVL is encoded by a V kappa III-segment most closely homologous to the Humhv328/L16 germline gene, whereas previous reports suggested V kappa III-encoded anti-Hib PS Ab might be exclusively encoded by the germline gene Humhv325/A27.(ABSTRACT TRUNCATED AT 250 WORDS)

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The V H3 associated RF idiotypes, D12 and B6, were expressed by 0 16 (0%) of monospecific RFs compared with 6 22 (27%) of polyreactive RFs. The predominant use of V H3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

Selection of immunoglobulin variable regions in autoimmunity to DNA.

Results from our analyses of variable region gene usage among spontaneous anti-DNA antibodies in autoimmune mice have indicated that both the early IgM and later-appearing IgG autoantibodies to DNA are generated by clonally selected B cells. The recurrent usage of particular variable region genes among all the anti-DNA hybridomas analyzed and reported to date supports this hypothesis. The preferential expression of particular light and heavy chain variable region genes among selected populations of both IgM and IgG anti-DNA hybridomas likewise supports the hypothesis. Both IgM and IgG antibody-producing B cells are derived from the same clonal precursor population and may be derived from the same B cell clonal precursor within an individual mouse. The selective and recurrent expression of germline and somatically-derived structures that would be expected to promote protein binding to DNA within anti-DNA antibody variable regions, particularly arginines in both light and heavy chain complementarity-determining regions, indicates that DNA or DNA-containing complexes may be the antigen that stimulates anti-DNA antibody in autoimmune mice. The progressive increase in the specificity of spontaneous anti-DNA antibodies for native DNA as the autoimmune response matures from IgM to IgG likewise suggests that DNA may be the antigenic stimulus for spontaneous anti-DNA in autoimmune mice. A hypothetical, computer-generated model of anti-DNA antibody binding to DNA provides an interesting paradigm for the molecular basis of antibody specificity for DNA.

T cell activation and T cell receptor variable region gene usage in measles.

T cell activation and T cell receptor variable (V) regions were studied with monoclonal antibodies in peripheral blood lymphocytes from 22 patients with measles. Increased (greater than 5%) activated T cells (HLA-DR+ CD3+ cells) were noted in 14 of the 22 patients. Elevations of V beta 5+ and V beta 8+ T cells were observed in two and four patients, respectively, and appeared to be associated with T cell activation. The duration of fever was significantly prolonged in those with increased (greater than 10%) activated T cells (p less than 0.01). These results suggest that T cell activation and the preferential expansion of V beta 8+ and V beta 5+ T cells are associated with the pathogenic process of measles.

Immunoglobulin light chain variable region gene sequences for human antibodies to Haemophilus influenzae type b capsular polysaccharide are dominated by a limited number of V kappa and V lambda segments and VJ combinations.

The immune repertoire to Haemophilus influenzae type b capsular polysaccharide (Hib PS) appears to be dominated by certain light chain variable region genes (IgVL). In order to examine the molecular basis underlying light chain bias, IgVL genes have been cloned from a panel of heterohybridomas secreting human anti-Hib PS (antibody) (anti-Hib PS Ab). One hybridoma, representative of the predominant serum clonotype of anti-Hib PS Ab in older children and adults following immunization or Hib infection, uses a V kappa II segment identical to the germline gene A2, and a JK3 segment. A second kappa hybridoma uses a member of the V kappa I family and a JK4 segment. Four lambda antibodies, all cross-reactive with the structurally related antigen Escherichia coli K100 PS, use V lambda VII segments which are 96-98% homologous to one another, and may originate from a single germline gene. Two additional lambda antibodies, not K100-cross-reactive, are encoded by members of the V lambda II family. All lambda antibodies use highly homologous J lambda 2 or J lambda 3 segments. The VJ joints of all lambda antibodies and the V kappa II-encoded antibody are notable for the presence of an arginine codon, suggesting an important role in antigen binding. Although more complex than heavy chain variable region gene usage, a significant portion of serum anti-Hib PS Ab is likely to be encoded by a limited number of V kappa and V lambda segments and VJ combinations, which may be selectively expressed during development, or following antigen exposure.

Mouse Vk gene classification by nucleic acid sequence similarity.

Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates ofV gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i. e., sequence similarity) and their organization in gene families. While mouseIgh heavy chainV region (V H) gene families are relatively well-established, a corresponding systematic classification ofIgk light chainV region (V k) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of theV k germline gene repertoire andV k gene usage in a variety of responses to foreign and self antigens, provides a classification of mouseV k genes in gene families composed of members with >80% overall nucleic acid sequence similarity. This classification differed in several aspects from that ofV H genes: only someV k gene families were as clearly separated (by >25% sequence dissimilarity) as typicalV H gene families; mostV k gene families were closely related and, in several instances, members from different families were very similar (>80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications forV k gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization betweenV H andV k genes.

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline

Predominant variable region gene usage by gamma/delta T cell receptor-bearing cells in the adult thymus.

Previous studies have indicated that the diversity of gamma genes expressed by gamma/delta-bearing murine T cells is limited, but comparable information concerning the expressed diversity of delta genes is lacking. In this study, we have investigated the rearrangement and expression of delta and gamma genes in T cell hybridomas that express gamma/delta T cell receptors. Three productive delta chain cDNA clones were isolated (delta 7.3, delta 7.1, and delta 2.3) that encode new variable region sequences. Two of the delta cDNAs differ significantly from those observed in the V alpha repertoire. In addition, one cDNA expressed a new J delta region (J delta 2), which was localized between J delta 1 and C delta genes. Using these and other delta gene probes and gamma gene probes, we found that five independent hybridomas expressed four different V delta s and three different V gamma s. However, analysis of an enriched population of gamma/delta-expressing cells from the adult thymus suggests that only a few V delta genes and one V gamma gene are used by the majority of the cells. These results suggest that important components of receptor chain that contribute to specificity (i.e., the germline V gene sequences) are relatively nondiverse in the thymic gamma/delta population.

Beta-chain gene rearrangements and V beta gene usage in DPw2-specific T cells.

The diversity of human T cell receptor beta-chain gene rearrangements and variable region gene usage in the T cell response to a single allogeneic class II HLA gene product has been investigated. Nine clones of cytotoxic T lymphocytes (8.2 to 8.10) that are specific for the class II specificity DPw2 were analyzed for their T cell receptor beta-chain gene rearrangements using a constant-region probe. A minimum of seven different clonotypes were present in this panel of clones. The beta-gene expressed by one clone, 8.9, was isolated and the variable (V), diversity (D), and joining (J) segments were sequenced. The sequence of the V beta 8.9 segment is identical to the V beta 14 sequence, and is joined to D beta 1.1 and J beta 1.1 segments. Northern analysis revealed that three of the eight clones analyzed, 8.5, 8.7, and 8.9, expressed a 1.3 kb transcript that hybridized with the V beta 8.9 probe, indicating that these clones were using the same V beta gene (these clones shared common DNA rearrangements). The remaining five clones did not express V beta 8.9. Southern analysis of DNA obtained from a DPw2-specific tertiary mixed lymphocyte reaction bulk culture from which the clones were derived showed a prominent rearrangement of the V beta 8.9 gene that was indistinguishable from those observed for clones 8.5, 8.7, and 8.9. This prominent rearrangement of V beta 8.9 was not observed in DNA obtained from normal peripheral blood lymphocytes. These results suggest that although the number of V beta genes which can contribute to a DPw2 specificity may be relatively large, only a limited number of clonotypes ultimately predominate in the response to certain class II HLA antigens.

T-cell receptor variable region gene usage in T-cell populations

T-cell receptor variable region gene usage in T-cell populations

T-cell receptor variable region gene usage in T-cell populations.

We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens.