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Human-human B-cell hybridomas were established using peripheral blood lymphocytes from patients with autoimmune thyroiditis and Graves' disease. Peripheral mononuclear cells (PMC), with or without mitogen prestimulation, were fused with HGPRT-negative human myeloma cell lines (Gm4672 and GM0462) using 44% polyethylene glycol. Developing hybridomas were screened by enzyme-linked immunosorbent assays (ELISAs) for human IgG and IgM and antibodies to human thyroglobulin (hTg) and microsomal antigen (M-Ag). A 125I-TSH binding inhibition assay was utilized for detecting antibodies to TSH receptor (TSH-R) protein. Hybridoma formation was observed only after prior mitogen stimulation of PMC. The amount of antibody secreted by the human-human hybridomas was highly variable (10 ng-100 micrograms/ml IgG/IgM). Nine and six-tenths percent of the hybrids secreted anti-hTg and 8.4% secreted anti-M-Ag. A 5% cloning efficiency was achieved, with detection of specific thyroid autoantibody secretion in one-third of the clones derived from positive hybridomas. Immunoglobulin secretion decreased with time and long-term stable clones were not achieved. Thyroid monoclonal autoantibodies to hTg, M-Ag, and TSH-R (IgG and IgM) detected during these studies were of a low affinity. In addition, antibodies were identified which exhibited marked specificity crossover between hTg, M-Ag, and nonthyroid antigens, suggesting the presence of recurrent epitopes. Such observations may help explain the multiplicity of thyroid autoantibodies in human thyroid disease and indicate a common defect in immunoregulation. We suggest that cross-reacting epitopes may be important in the derivation of thyroid-specific B-cell clones.

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The ⁵¹Cr cytotoxicity assay was modified for ethylnitrosourea (ENU) and methylnitrosourea (MNU) induced rat neurogenic tumor cell lines. Investigations on ‘immune’ lymphoid cytotoxicity showed little cytotoxicity above normal. Alteration in the level of lymphocytotoxicity was seen immediately after injection of a highly immunogenic tumor (EA-528). Natural cytotoxicity (NC) was present against all neurogenic tumor cell lines tested and xenogenic cell lines. The level of NC activity was dependent on age, specific tumor cell line and could be increased by isolating the nylon wool non-adherent ‘T’ cell population. Allogenic WFu rat lymphocytes were tested for NC activity in vitro against CDF tumor cells. NC activity has potential use against these tumors in vivo.

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