Pojen P. Chen

Molecular basis of an autoantibody-associated restriction fragment length polymorphism that confers susceptibility to autoimmune diseases.

Recently, combined serological and molecular studies of autoantibodies have revealed that these antibodies play an important role in the normal function of the immune system and in the development of the B cell repertoire. Accordingly, we hypothesized that a homozygous deletion of a critical autoantibody-associated Ig variable (V) gene may alter the immune system and thus predispose the host to autoimmune disorders. Initial experiments revealed several restriction fragment length polymorphisms (RFLP) of the Humhv3005 gene, that is likely to encode heavy chains of rheumatoid factors, and the closely related 1.9III gene. By probing EcoR1-digested DNA with the Humhv3005/P1 probe, we found that one of the four major hybridizing bands was missing in approximately 20% of patients with either rheumatoid arthritis or systemic lupus erythematosus, but only 2% of normal subjects. To delineate the genetic basis of this polymorphism, we have now employed the PCR to amplify and analyze hv3005, 1.9III, and homologous genes in individuals with characteristic RFLP genotypes. Our results indicate that the human Vh gene repertoire contains several hv3005- and 1.9III-like genes, and that a complete deletion of the hv3005-like genes is relatively restricted to a subset of autoimmune patients. These findings provide initial evidence for deletion of developmentally regulated autoreactive V genes in autoimmune diseases.

A natural autoantibody is encoded by germline heavy and lambda light chain variable region genes without somatic mutation.

While nonmutated germline variable region (V) genes have been found to encode heavy or light chains of various human autoantibodies, the use of germline V genes by both chains of a given autoantibody has not been documented. Recently, we reported that the heavy chain V gene (designated Humha346) of the Kim4.6 anti-DNA antibody is identical to a germline VH gene, 1.9III. To investigate whether this autoantibody was entirely germline encoded, we searched for the germline counterpart to the Kim4.6 V lambda segment (designated Humla146) and isolated a V lambda I gene designated Humlv117, which was identical to Humla146. Together with the sequence identity of the Kim4.6/Humha346 and 1.9III VH genes, the current data provide the first direct proof that an autoantibody can be encoded entirely by germline V genes without any somatic change. In addition, Humlv117 is the first V lambda I germline gene that has been isolated, and is highly homologous to the V lambda genes expressed in two lymphomas. Thus, this V lambda I gene should provide a useful tool for investigating the expression of the human V lambda gene repertoire, particularly with regard to autoimmune and/or lymphoproliferative diseases.

Anticardiolipin Antibodies From Patients With the Antiphospholipid Antibody Syndrome Recognize Epitopes in Both β2-Glycoprotein 1 and Oxidized Low-Density Lipoprotein

Background —We recently suggested that many anticardiolipin antibodies bind only to oxidized cardiolipin (OxCL) and/or to OxCL–&bgr;2-glycoprotein 1 (&bgr;2GP1) adducts but not to a “reduced” cardiolipin that is unable to undergo oxidation. To test this hypothesis, we investigated 24 sera, 4 protein A–purified IgG fractions, and 3 human monoclonal antibodies that were all isolated from patients with antiphospholipid antibody syndrome (APS); testing was also performed in 7 controls. Two monoclonal antibodies (IS3 and IS4) were selected for binding to CL and one was selected for binding to &bgr;2GP1 (LJB8). Methods and Results —By chemiluminescent immunoassay, all APS sera samples bound only to OxCL and not to reduced CL, and the binding was inhibited >95% by OxCL but not reduced CL. All purified IgG fractions bound to &bgr;2GP1 but only when the &bgr;2GP1 was plated on microtiter wells coated with OxCL. All 3 monoclonal antibodies bound only to OxCL. On Western blots, IS4 and LJB8 bound to &bgr;2GP1 as well as to delipidated apoB of oxidized LDL but not to native apoB. IS3 also bound to oxidized apoB on Western blot. Covalent modification of &bgr;2GP1 with oxidation products of CL made it more antigenic for APS serum samples, for purified IgG fractions, and for the monoclonal antibodies. Conclusions —These data support the hypothesis that oxidation of CL is needed to generate epitopes for many anticardiolipin antibodies and that some of these epitopes are covalent adducts of OxCL with &bgr;2GP1 or apoB.

Structural Analyses of Human Developmentally Regulated Vh3 Genes

In mice, a restricted set of the Jh‐proximal Vh genes are preferentially expressed during early ontogeny. Recently, analyses of human Ig cDNA from a fetal liver revealed a restricted set of Vh genes which belong to the VhI, 3, 4, and 6 families. Although the Vh6 and some Vh5 genes are proximal to the Jh region, no Vh5 gene was found in the fetal liver, suggesting that the distance between the Jh genes and some early‐expressed Vh genes may not be the only factor responsible for Vh gene expression during early development. As an initial step in searching for other underlying mechanisms, we characterized two human germline Vh3 genes which belong to the developmentally restricted Vh repertoire, and found that they contain many enhancer‐like sequences which are identical, or highly homologous to, various transcriptional enhancer motifs. Hence, it is conceivable that, in addition to the established positional effects, cis regulatory elements may be important in the programmed expression of some Vh genes during early B‐lymphocyte development.

The incidence of a new human cross-reactive idiotype linked to subgroup VHIII heavy chains

Cross-reactive idiotypes (CRI) on human rheumatoid factors (RF), which are identified by murine monoclonal antibodies (mAb), have proved useful in defining both the incidence and the structural characteristics of these autoantibodies. In this study, a new murine anti-idiotypic reagent, mAb B6, has been used to identify and define the expression of a distinct heavy chain CRI. The B6 CRI was found on 20% of monoclonal IgM (16 of 81), but on only 5% of monoclonal IgA (1 of 20) and on no monoclonal IgG. In addition, this CRI was expressed exclusively on a subset of Ig derived from the VHIII protein variable region subgroup. In immunoblotting experiments, the mAb B6 bound directly to the heavy (H) chains of CRI positive proteins. The B6 CRI was found frequently on monoclonal IgM-RF molecules, and the mAb B6 could inhibit the binding of the RF to its IgG antigen. It was also demonstrated that Staphylococcus aureus protein A (SpA), which has recently been shown to bind to the F(ab) region of VHIII molecules, could block the interaction of some B6 CRI positive IgM to the anti-CRI. These experiments suggest that the B6 CRI is a marker for one or a few VHIII genes and that it is expressed commonly on IgM paraproteins, many of which have RF activity.

A Human Anti-Cardiolipin Autoantibody is Encoded by Developmentally restricted Heavy and Light Chain Variable Region Genes

Based on recent structural analyses of monoclonal autoantibodies, it appears that a number of these antibodies express germ-line immunoglobulin variable region (V) genes with little or no somatic mutation. In addition, our group and others have noted the identity or near identity of some autoantibody-associated V genes to V genes apparently expressed preferentially in the fetal pre-B cell repertoire. To extend these data, we now report that the heavy and light chain V genes of an anti-cardiolipin antibody derived from a healthy individual display 99% nucleotide sequence homology with V genes expressed in early B cell ontogeny. Sequence comparisons indicate the likely use of fetal-restricted V genes by this autoantibody. Taken together with other data on autoantibody V gene usage, these findings provide further evidence for overlap between the autoantibody-associated and early ontogeny expressed V gene repertoires and suggest that natural autoreactivity may be instrumental in the development and maintenance of the normal immune repertoire.

The human immunoglobulin V(H) gene repertoire is genetically controlled and unaltered by chronic autoimmune stimulation.

The factors controlling immunoglobulin (Ig) gene repertoire formation are poorly understood. Studies on monozygotic twins have helped discern the contributions of genetic versus environmental factors on expressed traits. In the present experiments, we applied a novel anchored PCR-ELISA system to compare the heavy chain V gene (V(H)) subgroup repertoires of mu and gamma expressing B lymphocytes from ten pairs of adult monozygotic twins, including eight pairs who are concordant or discordant for rheumatoid arthritis. The results disclosed that the relative expression of each Ig V(H) gene subgroup is not precisely proportional to its relative genomic size. The monozygotic twins had more similar IgM V(H) gene repertoires than did unrelated subjects. Moreover, monozygotic twins who are discordant for RA also use highly similar IgM V(H) gene-subgroup repertoires. Finally, the V(H) gene repertoire remained stable over time. Collectively, these data reveal that genetic factors predominantly control V(H) gene repertoire formation.

Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors.

A major diagnostic marker in most rheumatoid arthritis (RA) patients is the rheumatoid factor (RF), an autoantibody that binds to the Fc region of IgG. To delineate the Ig genes and the underlying mechanism for RF production in RA patients, we applied a systematic approach to define the genetic origins of three IgG RFs derived from the synovial fluid of two RA patients. The results show that two of three IgG RF have substantial numbers of somatic mutations in their variable (V) regions, ranging from 13 to 23 mutations over a stretch of 291-313 nucleotides, resulting in a frequency of 4.4-7.8%. However, one IgG RF has only one mutation in each V region. This result indicates that an IgG RF may arise from a germline gene by very few mutations. The mutations occur mainly in the complementarity-determining regions (CDRs), and the mutations in the CDRs often lead to amino acid substitutions. Five of the six corresponding germline V genes have been found to encode either natural autoantibodies or autoantibodies in other autoimmune disorders; and three of the six V genes have been found in fetal liver. Taken together with other results, the data show that (a) several potentially pathogenic RFs in RA patients arise from natural autoantibodies, and (b) only a few mutations are required to convert the natural autoantibodies to IgG RFs.

Molecular characterization of the human immunoglobulin VλI germline gene repertoire

Abstract To advance our understanding of the human immunoglobulin Vλ germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the VλI subgroup. These genes can be divided into three sub-subgroups on the basis of ⩾ 93% nucleotide sequence homology and ⩾ 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed VλI genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed Vλ I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other VλI germline genes (Humlv 101 and Humlv 1041) which may interfere with their expression.

Molecular analysis of human immunoglobulin Vλ germline genes: Subgroups VλIII and VλIV

Abstract Three human immunoglobulin Vλ germline genes have been isolated: two from the VλIV subgroup and one from the VλIII subgroup. The VλIII gene and one of the VλIV genes appear to be functional (each being utilized in at least two expressed Vλ genes), despite deviations from the reported consensus sequences in their promoter TATA-box and recombination signal sequence elements. The other VλIV gene is a pseudogene. Of the 20 human Vλ germline genes characterized to date, 45% are pseudogenes or vestigial genes.