VH4-21, a human VH gene segment overrepresented in the autoimmune repertoire.
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Autoimmunity↗
Biomedical subjects
J D Capra
Publications and source records attributed to J D Capra.
Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.
A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.
Adult↗
Human monoclonal striational autoantibodies isolated from thymic B lymphocytes of patients with myasthenia gravis use VH and VL gene segments associated with the autoimmune repertoire.
The production of autoantibodies reactive with components of skeletal muscle is characteristic of myasthenia gravis (MG) and is strongly associated with the presence of thymic epithelial tumors in patients with MG. The nucleotide sequences of the heavy and light chain variable regions (VH and VL) of three human monoclonal striated muscle antibodies (StrAb) isolated from thymic B lymphocyte lines from two patients with MG and thymoma were analyzed. The VH and VL gene segments used by these anti-striated muscle antibodies appear to be derived from the same repertoire of gene segments as have been found in other autoantibodies isolated from patients with a number of different autoimmune diseases. While the IgM StrAb SA-1A is a direct copy of germ-line VH and VL gene segments, analysis of the IgG StrAb SA-4A and SA-4B, which may be more representative of antibodies found in the serum of MG patients, suggests that the processes of antigenic selection and somatic mutation may contribute to the generation of autoantibodies in MG.
Actins↗
Human anti-acetylcholine receptor antibodies use variable gene segments analogous to those used in autoantibodies of various specificities.
The production of autoantibodies to the nicotinic acetylcholine receptor are responsible for many of the neurological symptoms observed in myasthenia gravis. An understanding of the structural organization of the anti-receptor antibodies may help to define the role of these antibodies in the pathogenesis of this disease. The nucleotide sequences of the heavy and light chains of three human monoclonal anti-receptor antibodies isolated from peripheral blood lymphocytes from two patients with myasthenia gravis were analyzed. In addition, the structure of an anti-idiotypic antibody was studied. The VH and VL gene segments used in the anti-receptor antibodies appear to be derived from the same repertoire as gene segments that have been found in other autoantibodies isolated from patients with various autoimmune diseases. The IgM anti-receptor antibodies are direct copies of germline gene segments, while the structures of the IgG anti-receptor antibody and the anti-idiotypic antibody appear to be mutated suggesting that they have undergone antigenic selection.
Amino Acid Sequence↗
Analysis of HLA genotypes and susceptibility to insulin-dependent diabetes mellitus: HLA-DQ alpha complements HLA-DQ beta.
It is well known that certain genes in the HLA-D region confer increased susceptibility to insulin-dependent diabetes mellitus (IDDM). Previous studies have documented an increased risk associated with the HLA-DR beta chain alleles, DR3 and DR4, and the DQ beta chain allele DQB1*0302 (formerly DQw8). Since DQ alpha is also polymorphic and has been strongly implicated as the primary IDDM susceptibility locus in other races, we wanted to assess the contribution of DQ alpha to IDDM in Caucasians. This information would enable us to define more precisely the class II association with IDDM as well as gain insight into issues of cis versus trans association of DQ heterodimers in this disease. To this end, the DQ alpha genotype was determined for a large group of diabetic and normal Caucasian individuals who had been HLA-DQ beta and HLA-DR typed previously. Using the polymerase chain reaction and a set of twelve oligonucleotide probes, we determined the DQ alpha genotype of 323 patients with IDDM and 182 normal subjects. We found that certain DQ alpha alleles are decreased in the diabetic population compared with normal subjects (i.e. DQA1*0102 and *0103), while others are significantly increased in patients with IDDM (i.e. DQA1*0301 and *0501). In addition, certain combinations of DQ alpha alleles are associated with increased susceptibility to disease (i.e. DQA1*0301, *0501). These results parallel our findings at the DQ beta locus; however, because of the various associations between DQ alpha and DQ beta chains, the risks conferred by DQ alpha are generally lower than those at DQ beta. Moreover, our data indicate that, in Caucasians, no single DQ alpha allele accounts for the highest degree of susceptibility to IDDM as in other races, although DQ alpha analysis may be informative in a few cases. When done in combination, however, oligonucleotide analyses at both DQ alpha and DQ beta complement each other and provide a more complete assessment of the HLA-associated component of disease susceptibility in IDDM.
Alleles↗
Analysis of HLA genotypes and susceptibility to insulin-dependent diabetes mellitus: association maps telomeric to HLA-DP.
There is convincing evidence that certain combinations of alleles within the human leucocyte antigen (HLA) complex, particularly within HLA-DQ, are associated with either resistance or susceptibility to insulin-dependent diabetes mellitus (IDDM). A previous study conducted on a large, well-defined group of patients demonstrated that DQB1*0302 (DQw8) conferred 'dominant susceptibility' to IDDM while DQB1*0602 (DQw1.2) conferred 'dominant protection'. The availability of this population enabled us to further assess susceptibility associated with other class II alleles in an effort to map an outside HLA boundary of disease association. Using a group-specific polymerase chain reaction protocol and a series of oligonucleotide probes which define over twenty DP beta alleles, we studied 286 unrelated Caucasian patients with IDDM and 184 normal subjects. We found that while several alleles are increased (DPB1*0201, DPB1*0301, DPB1*0402) or decreased (DPB1*0101, DPB1*0202) in the diabetic population compared with the normal subjects, the HLA association with IDDM is considerably weaker at the DP locus. These data define the centromeric boundary for the HLA-associated susceptibility gene in IDDM, localizing susceptibility to the region telomeric to HLA-DP up to and including HLA-DQ.
Adolescent↗
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 VH and VL 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 VH3 genes beyond that one would expect based on random utilization.
Amino Acid Sequence↗
Rheumatoid factor V genes from patients with rheumatoid arthritis are diverse and show evidence of an antigen-driven response.
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Amino Acid Sequence↗
Serological cross-reactivity between a human Ro/SS-A autoantigen (calreticulin) and the lambda Ral-1 antigen of Onchocerca volvulus.
We have cloned and sequenced a 46-kD Ro/SS-A autoantigen gene that is the human homologue of the calcium-binding protein, calreticulin. The sequence of this 46-kD Ro/SS-A protein (calreticulin) has significant homology to lambda Ral-1, a recombinant cDNA clone corresponding to a major antigen of the nematode, Onchocerca volvulus, the infectious agent of onchocerciasis. We therefore sought to determine whether antibodies produced by onchocerciasis patients might crossreact with the human 46-kD Ro/SS-A autoantigen (calreticulin). 20 of 22 sera from Liberian onchocerciasis patients who had no known evidence of autoimmune disease were found to contain antibodies that reacted with the 46-kD Ro/SS-A (calreticulin) by immunoblot analysis. Characteristic of sera reactive with Ro/SS-A antigens, some onchocerciasis sera also immunoprecipitated the Ro/SS-A-associated hY RNAs. In addition, a monoclonal antibody raised against O. volvulus organisms reacted to purified human WiL-2 cell 46 kD Ro/SS-A antigen (calreticulin) by ELISA. These results strongly suggest that onchocerciasis patients produce antibodies that crossreact with the 46-kD human Ro/SS-A autoantigen (calreticulin) and raise the possibility that infectious organisms such as O. volvulus might play a triggering or exacerbating role in the human Ro/SS-A autoimmune response.
Amino Acid Sequence↗
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 VH gene segments provides the bulk of the immunoglobulin heavy chain repertoire. Most of these VH 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 VH families, and more specifically one single VH gene contributes to a large extent to the adult autoimmune repertoire in restricted as well as unrestricted responses. This VH gene segment is not particularly overexpressed in the fetus. Since the only common element to these autoreactive responses is the region encoded by the VH gene itself, these observations may provide an important insight into B-cell regulation.
Adult↗
IgM rheumatoid factors in patients with rheumatoid arthritis derive from a diverse array of germline immunoglobulin genes and display little evidence of somatic variation.
Rheumatoid factors (RF) are present in the plasma of patients with rheumatoid arthritis (RA) although the site of synthesis of most of these antibodies is within the synovium. This report primary concerns RF of the IgM isotype. While a few of the RF derive from patients with systemic lupus erythematosus or from normal individuals, the remaining derive from the inflamed synovial tissue of patients with RA. Two RF are encoded by members of the VH1 gene family, 8 from the VH3 family and 2 from the VH4 family. Two polyreactive antibodies derive from the VH3 family and 2 come from the VH4 family. This distribution is not fundamentally different from the distributions seen in a large array of autoantibodies and antibodies to external antigens. Similarly, the light chains derive from most of the known kappa and lambda VL families. It is hard to escape the preliminary conclusion that gene segments from virtually any light chain variable region can contribute to RF or polyreactive antibody structures. Most IgM RF and polyreactive antibodies are direct copies of germline genes in one of their polypeptide chains or at most are 2 nucleotides away in one of their chains from a known germline gene.
Antibody Formation↗
Mutational analysis of the cross-reactive idiotype of the A strain mouse.
The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each of several monoclonal idiotopes has allowed us to identify more precisely the amino acids responsible for expression of the CRIA. Thus we have shown that expression of the CRIA is equally dependent on amino acids in the second and third CDR of the H chain. Furthermore, the idiotopes studied surround the Ag-binding site and clearly involve multiple interactions in several of the L and H chain CDR.
Amino Acid Sequence↗
Molecular characterization of five human anti-human immunodeficiency virus type 1 antibody heavy chains reveals extensive somatic mutation typical of an antigen-driven immune response.
We report the heavy chain variable region sequences from the cDNAs of five previously described monoclonal cell lines producing human antibodies specific for the human immunodeficiency virus type 1 and detail the molecular characteristics, germ-line origins, and extent of somatic mutation among these antibodies. Three of the five heavy chain variable regions derive from the VHIV gene family, but each has arisen from a different heavy chain variable region (VH) gene segment within the VHIV family. In addition, one is derived from a VHI gene segment, and one is derived from a VHV gene segment. Since four of the five antibodies arise from known germ-line VH elements, a precise determination of the extent of somatic variation is possible. The amount of variation from the closest germ-line sequence ranges from 4.5% to 14.8% among these antibodies, most of which is concentrated in the complementarity-determining regions. In general, the diversity (D) segments are long, characteristic of D-D fusions and/or extensive terminal deoxynucleotidyltransferase activity; however, definitive homologies cannot be found with the known germ-line D segments. Joining (JH) gene segment utilization appears random. The use of five different germ-line VH gene segments and extensive somatic mutation provides evidence that a polyclonal, antigen-driven immune response occurs during the natural infection with human immunodeficiency virus.
Amino Acid Sequence↗
Nucleotide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype.
Cold agglutinins are human autoantibodies, usually of the IgM class, which agglutinate RBC at low temperature. The major subset recognizes the I/i carbohydrate Ag, and many of these antibodies bear cross-reacting idiotypic determinants. An anti-idiotypic mAb that is specific for one of the idiotopes largely confined to cold agglutinins has been used to identify and monitor tumor cells that secrete these molecules in two patients. The tumor cells were immortalized with EBV and the idiotope-positive lines used to investigate the utilization of the VH and VL genes by these antibodies. Nucleotide sequence analysis of the two cold agglutinins (FS-1 and FS-2) revealed the utilization of a single common gene segment, VH4-21. Serologic analysis documented that only human antibodies utilizing the VH4-21 gene segment were reactive in the idiotope assay, other VHIV antibodies as well as a panel of antibodies derived from other VH families being negative. The DH, JH, VK, and JK gene segments of FS-1 and FS-2 were structurally distinct. These data suggest that the structural basis for the cross-reactive idiotope as well as cold agglutinin activity is the VH4-21 gene segment. A nucleotide change in H chain CDR1 of both cold agglutinins results in the substitution of an aspartic acid residue for glycine at position 31, suggesting that this amino acid might be critical to recognition of the red cell Ag.
Aged↗
Mutational analysis of arsonate binding by a CRIA+ antibody. VH and VL junctional diversity are essential for binding activity.
To assess the impact of various heavy and light chain mutations on p-azophenylarsonate binding, murine antibodies have been produced in insect cells (SF9) utilizing a baculovirus expression system. When expressed in this system, an antibody composed of a canonical CRIA+ heavy and light chain can bind antigen and express idiotype indistinguishably from analogous hybridoma-derived antibodies. Antibodies comprised of either light chains mutant at the V-J junction or heavy chains mutant at the V-D junction were found to be incapable of binding arsonate. In addition, substitutions in the first and second complementarity determining regions of the heavy chain were shown to play a role in arsonate binding, most likely related to affinity maturation targeted at the carrier protein. These results confirm the obligatory role that junctional diversity plays in the generation of arsonate-specific antibodies, as well as extend our understanding of the role of other variable region amino acids in arsonate binding.
Amino Acid Sequence↗
Conservation of the most JH proximal Ig VH gene segment (VHVI) throughout primate evolution.
The human VHVI gene segment, the sole member of the VHVI gene family, is remarkable in that it is the most D-proximal VH gene segment and is apparently nonpolymorphic. Here we report that the VHVI gene segment has been remarkably preserved in primate evolution. We were unable to detect RFLP among several primates, and nucleotide sequences of several VHVI gene segments showed remarkable conservation. No differences were detected in the nucleotide sequences of the VHVI gene segment from three unrelated chimpanzees. These findings suggest that the VHVI gene segment has been strongly selected for during primate evolution, suggesting an important immunologic role.
Amino Acid Sequence↗
Polymorphism in V kappa 10 genes encoding L chains of antibodies bearing the Ars-A and A48 cross-reactive idiotypes.
p-azophenylarsonate-specific antibodies of A/J mice which bear the Ars-A crossreactive idiotype utilize the V kappa-Ars-A gene segment, a member of the V kappa 10 family. Southern hybridization of genomic DNA from several inbred strains using a probe from the 5' flanking region of the V kappa-Ars-A gene demonstrated three patterns of restriction fragment length polymorphisms (RFLP). Six genes corresponding to hybridizing bands were obtained from DNA libraries of C.AKR, PERU and A/J mice, and nucleotide sequence comparisons revealed two allelic groups: AKR1 (Igk-V10.1a), AJ1 (Igk-V10.1b) and PERU1 (Igk-V10.1c); AKR2 (Igk-V10.2a), AJ2 (Igk-V10.2b), and PERU2 (Igk-V10.2c). The Igk-V10.1b gene of the A/J strain is the V kappa-Ars-A gene used in Ars-A idiotype-positive antibodies. The product of the C.AKR allele (Igk-V10.1a) contained four amino acid substitutions in CDR3 as compared with Igk-V10.1b. These substitutions probably explain the failure of AKR mice and other strains with the same V kappa 10 RFLP pattern to provide in genetic crosses a L chain which, together with the A/J VH-ArsA gene product, form Ars-A idiotype-positive antibodies. Also, the nucleotide sequence identity between the Igk-V10.1c and Igk-V10.1b alleles and the Igk-V10.2c and Igk-V10.2b alleles is significantly greater than that seen in comparisons with the Igk-V10.1a and Igk-V10.2a alleles, respectively, suggesting an evolutionary pathway similar to that of the linked Igk-J locus. BALB/c antibodies bearing the A48 regulatory idiotype contain L chains encoded by the BALB/c Igk-V10.1b and Igk-V10.2b alleles. Strongly A48 idiotype-positive antibodies utilize the Igk-V10.1b chain, and weakly A48-positive antibodies use the Igk-V10.2b L chain. The possible effects of amino acid substitutions specified by the Igk-V10.1a, Igk-V10.1c, Igk-V10.2a, and Igk-V10.2c alleles on their ability to provide L chains used in A48 idiotype-positive antibodies are discussed.
Alleles↗