J D Capra

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 Sjogrens 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.

Monoclonal antibodies against human monocytes. II. Recognition of two distinct cell surface molecules

Three monoclonal antibodies (63D3, 63D2, and 61D3) with reactivity against human monocytes have been studied. They reacted with most adherent mononuclear cells but not with pure populations of peripheral blood B and T lymphocytes. Two of these antibodies, 63D3 and 63D2, competed in fluorescence inhibition experiments, recognized a monocyte surface antigem of about 200,000 daltons, and werre idiotypically related. The third antibody, 61D3, did not compete with the others in fluorescence inhibition experiments, recognized a different surface molecule, and was idiotypically distinct from 63D3 and 63D2. Whereas 63D3 and 63D2 reacted very weakly with some granulucytes, 61D3 did not, suggesting that it is specific for monocytes alone.

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 VH4 encoded antibodies. 9G4 exclusively binds VH4–21 encoded antibodies, while LCI binds antibodies derived from VH4 family gene segments V71‐2, V71‐4, VH4‐18, VH72‐I and V2‐1. The VH4–21 gene segment is utilized by most cold agglutinin (CA) antibodies with I/i specificity, while antibodies encoded by other VH4 gene segments are associated not with CA disease, but primarily with rheumatoid‐factor (RF) activity.

4-Aminoquinoline antimalarials enhance UV-B induced c–jun transcriptional activation

Previous work has documented that the earliest observable response in mammalian cells following ultraviolet (UV) irradiation is the activation of plasma membrane-associated Src tyrosine kinases. These molecules then trigger a signalling cascade that results in activation of the transcription factor AP-1 which subsequently transactivates the early immediate genes including c–jun. This pathway has been postulated to play a protective role against UV damage. As aminoquinoline antimalarials such as chloroquine are known to downregulate several photoinduced cutaneous disorders including LE-specific skin disease, we asked whether chloroquine might be capable of modulating this early limb of the UV light response. A431 cells (a human epidermal keratinocyte cell line) that had been transfected with a c–jun luciferase reporter gene construct were then treated with physiologically relevant concentrations of chloroquine followed by exposure to 0—125 J/m2 of UV-B from a bank of unfiltered FS20 lamps. Chloroquine pretreatment resulted in a dose–dependent increase in luciferase activity in permanently transfected A431 cells (luciferase activity was increased by 45% at 2.5 × 10-5 M chloroquine and 125 J/m2 of UV-B). Hydroxychloroquine pretreatment also resulted in an increase in luciferase activity. Primaquine, an 8-aminoquinoline, did not influence the UV-B induced c-jun activity. Furthermore, chloroquine did not have a similar impact on HSP-70 gene activity during heat shock. These studies suggest that the beneficial effect of the 4-aminoquinoline antimalarials in various photodermatoses including cutaneous LE might result in part from the capacity of these drugs to enhance the protective early limb of the UV response.

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. The availability of a large population of healthy subjects and sequence‐specific oligonucleotide (SSO) typing enabled us to assess the degree of linkage between HLA‐DPBl and HLA‐DQBl genes. Using the polymerase chain reaction and a series of oligonucleotide probes which define seven DQβ alleles and twenty DPβ alleles, we studied 180 unrelated, normal Caucasian individuals and found only weak or negative associations between HLA‐DPBl and HLA‐DQBl. These data demonstrate that the association between HLA‐DQ and DP is weak and also imply that DP extended haplotypes related to particular diseases may not reflect normal associations. Implications of these results might impact on the concept of linkage disequilibrium in general as well as the evolution of the HLA complex. In addition, extensions of this work may have clinical ramifications with regard to bone marrow transplantation and founder effects in certain diseases.

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. To investigate the nature of the T‐cell response to BCG in RA, we isolated T cells from the synovial fluid of a patient with early‐stage rheumatoid arthritis, stimulated them with BCG and cloned by limiting dilution. Staining with monoclonal antibodies specific for different Vβ gene families revealed a statistically significant greater proportion of synovial‐derived T‐cell clones expressing the Vβ8 gene family product compared with peripheral blood clones. While the antigen specificity of some of the clones could not be determined, several of the clones displayed distinct antigen reactivities. Sequencing the TCR P chain genes of these T cells suggested that although the Vβ8 gene products appeared to be over‐represented in these BCG‐specific clones, each clone utilized distinct Jβ gene segments and used N segment addition to different extents. In addition, no common motifs were identified in the β chain CDR3s of the clones sequenced. Analysis of bulk cultured BCG‐specific SF T cells and unstimulated peripheral blood T cells for Vβ8 gene expression also revealed a large amount of diversity within the CDR3 region. Thus, the T‐lymphocyte response to BCG in this patient with early rheumatoid arthritis appears to be quite heterogeneous.

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 grou p of patients demonstrated that DQB 1*0302 (DQw8) conferred ‘dominant susceptibility’ to IDDM while DQB 1*0602 (DQwl.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 HL A boundary of disease association. Using a group‐specific polymerase chain reaction protocol and a series of oligonucleotide probes which define over twenty DPβ alleles, we studied 286 unrelated Caucasian patients with IDDM and 184 normal subjects.

Allorecognition of HLA DR2 and DR5 Molecules by V-Beta-8-Positive T-Cell Clones

V‐beta‐8‐positive T cells were isolated from human peripheral blood lymphocytes using a monoclonal antibody specific for the Vβ8 family. Alloreactive T‐cell lines were generated by stimulation with mononuclear cells from individuals homozygous for HLA DRI‐DR9. Cloned Vβ8‐positive T cells were then assayed for alloreactivity based on a proliferative assay using irradiated B‐cell lines. Vβ8‐positive T‐cell clones alloreactive to DR2 and DR5 molecules were chosen for further study based on the association of these MHC antigens with autoimmune disease. DNA sequence analysis confirmed the use of the Vβ8 gene family as well as providing information on the use of the V, D, J and N segments in these alloreactive T‐cell clones.

The MoAb-VκIIIb Cross-Reactive Idiotope on A27a (Humkv325) Encoded Kappa Chains Maps to Framework Region 3

The monoclonal antibody MoAb‐VκIIIb binds a cross‐reactive idiotopic (CRI) determinant on light (L) chains encoded by the VκIIIb subgroup A27a (Humkv325) gene segment. The aim of this study was to localize the MoAb‐VκIIIb CRI. Mutational analyses involving region exchanges between a CRI‐positive VκIIIb chain and a CRI‐negative Vκ1 chain indicate that the MoAb‐VκIIIb CRI is located in framework region (FR) 3 of A27a (Humkv325) encoded L chains. CRI‐positive kappa chains unpaired with a heavy (H) chain are reactive with MoAb‐VκIIIb, indicating that the CRI is located on the kappa chain alone without involvement of H chain residues. Combinatorial antibodies composed of non‐parental L and H chain pairings are reactive with MoAb‐VκIIIb only when the L chain is A27a (Humkv325) encoded. The CRI, therefore, is not readily perturbed by H chain interactions. When the FR3 from a CRI‐positive kappa chain replaced the FR3 in a CRI‐negative lambda chain, the determinant was no longer detectable with MoAb‐VκIIIb. It is possible, therefore, to exchange regions between kappa chains from different families and retain the CRI structure, however the determinant is lost when placed in a more foreign background such as a lambda chain. These data more precisely define the interaction between MoAb‐VκIIIb and its CRI, and indicate that there are limits within which antibody FRs can be shuffled and still retain their native structural features.