Hélène Massicotte

We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

Human hybridomas have been produced by fusing peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with the GM 4672 human cell line. 262 hybridoma clones from the fusions of four RA and five SLE patients were screened for binding to denatured DNA (dDNA), native DNA, and the Fc fragment of human IgG (HIgG). Of the 17 hybridoma antibodies (nine RA, eight SLE) selected for strong binding to denatured DNA, Fc, or both, five reacted with dDNA only, one with Fc only, and eight with both dDNA and Fc. Hybridoma supernatants exhibiting dual reactivity were absorbed over HIgG and bovine serum albumin (BSA)-Sepharose immunoabsorbent columns. The reactivities to both DNA and HIgG were completely removed by the HIgG column but unaffected by passage over the BSA column, and both DNA binding and rheumatoid factor activities were recovered in the acid eluates from the Sepharose-IgG column. The binding of dual reactive hybridoma autoantibodies to the Fc fragment of HIgG was specifically competed by dDNA and HIgG, providing additional evidence that one antibody may be capable of reacting both as a rheumatoid factor and as an anti-DNA antibody.

The utilization of one human lymphoblastoid cell line in fusion experiments with peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE) has made it possible to define efficient and reproducible conditions for the production of anti-DNA-secreting human-human hybridomas. This investigation, using the human lymphoblastoid cell line GM 4672 fused in the presence of 44% polyethylene glycol with lymphocytes from SLE patients, demonstrated a maximal yield of 22.8% hybridomas, 17% of which produced anti-DNA antibodies. We were able to define, in two independent laboratories, that the maximal yield of hybridomas occurred when the lymphocyte to GM 4672 cell ratio was 1:1 and cells were seeded in 2.0 ml wells at a concentration of 4 X 10(5) cells/well. This report demonstrates the reproducibility of human-human hybridoma production with the GM 4672 cell line and the establishment of efficient conditions for the production of anti-DNA autoantibodies from SLE patients.

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.