Heterogeneity of the TCR repertoire in synovial fluid T lymphocytes responding to BCG in a patient with early rheumatoid arthritis.

T lymphocytes have been implicated in the pathogenesis of rheumatoid arthritis. Interestingly, many of the activated T cells isolated from the synovial fluid of individuals with rheumatoid arthritis react with antigens from Mycobacterium tuberculosis or BCG. This response is seen to a much lesser extent in the peripheral blood of these patients.

Synovial IgG rheumatoid factors show evidence of an antigen-driven immune response and a shift in the V gene repertoire compared to IgM rheumatoid factors.

We have established IgG rheumatoid factor (RF)-secreting hybridoma cell lines from the synovial tissues of three patients from whom we have previously characterized several IgM RF. The IgG RF bind human and rabbit IgG and form intracellular complement-fixing complexes indicative of a self association process in vivo. Nucleotide sequence analysis revealed that two IgG RF used VHIII gene segments, while one used a VHI gene segment.

Nucleotide sequence of a human autoantibody to the alternative pathway C3/C5 convertase (C3NeF).

The production of autoantibodies to the alternative pathway C3/C5 convertase or C3 Nephritic Factor (C3NeF) is one characteristic of membranoproliferative glomerulonephritis. The complete nucleotide sequences of the heavy and light chain variable regions of an IgG C3NeF produced by an EBV transformed B cell line derived from a patient with membranoproliferative glomerulonephritis were determined. The VH and VL gene segments used by this C3NeF are extensively mutated suggesting that antigenic selection and affinity maturation may occur during the generation of these autoantibodies.

Construction of recombinant DNA by exonuclease recession.

We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions.

Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts.

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Linkage disequilibrium within the HLA complex does not extend into HLA-DP.

It is well known that certain alleles from different loci within the Human Leucocyte Antigen (HLA) complex are in linkage disequilibrium. This linkage phenomenon is relatively well characterized for haplotypes that include specific class I and class II alleles such as HLA-B8 and HLA-DR3. However, the HLA-DP genes are located at the centromeric end of the HLA complex and are less well characterized with regard to linkage disequilibrium.

Antibody production in the baculovirus expression system.

The proven ability of insect cells to produce murine and human antibodies renders the baculovirus system amenable to the synthesis of both naturally occurring antibodies and designed mutants. Study of these antibodies should provide a basis for rational antibody design useful in human therapy.

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4.

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.

VH restriction among human cold agglutinins. The VH4-21 gene segment is required to encode anti-I and anti-i specificities.

We previously reported that human autoantibodies with cold agglutinin activity contained a single human VH gene segment (VH4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To confirm and extend this observation we have analyzed at the nucleotide level the H and L chains of six new cold agglutinin molecules derived from different patients. We found that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the VH gene segment level is absolute.

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.

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.

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'.

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.

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).

Clonally related IgM rheumatoid factors undergo affinity maturation in the rheumatoid synovial tissue.

Using hybridoma technology we established a panel of human monoclonal rheumatoid factors (RF) from the synovial tissues of two patients with rheumatoid arthritis (RA), and one patient with polyarticular juvenile RA. Nucleotide sequence analysis of the V regions of these RF indicates that two independently derived antibodies from one of the RA patients are clonally related. One of these antibodies appears to be close to germ-line configuration, whereas the other has accumulated a total of 36 substitutions in both H and L chains.

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.

Anti-CD3-stimulated T cells induce the production of multiple Ig H chain isotypes by individual human peripheral B lymphocytes.

Polyclonal activation of human B cells is achieved by coculture with T cells stimulated by mAb to the CD3 molecular complex. By formal limiting dilution analysis, approximately 60% of human peripheral blood B cells were found to produce Ig in this system. When individual B cells were cultured in microtiter wells with anti-CD3-activated T cells, more than one-third of cultures producing Ig contained multiple Ig H chain isotypes.

The 5'-flanking region of the human calreticulin gene shares homology with the human GRP78, GRP94, and protein disulfide isomerase promoters.

Calreticulin (CR) is a calcium binding protein that resides in the endoplasmic and sarcoplasmic reticulum and is reactive with human Ro/SS-A autoimmune sera. We have used human CR cDNA to isolate a human 6-kilobase genomic clone that contains 529 base pairs upstream of the presumed transcription start site, 9 exons, 8 introns, and several hundred base pairs 3' of a polyadenylation sequence. Analysis of the human CR promoter region reveals a number of potential regulatory sites also found in the human GRP78, GRP94, and protein disulfide isomerase promoters, including multiple Sp1 and CCAAT consensus sequences, an AP-2 recognition sequence (absent in protein disulfide isomerase), and multiple GC-rich areas.

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.

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.

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.