Potential role of PHA in producing human monoclonal thyroid autoantibodies of different subclasses

A human monoclonal autoantibody to thyroglobulin (Tg) of subclass IgG2 was developed by fusing a mouse myeloma with Tg antibody secreting Epstein-Barr virus (EBV)-infected B lymphocytes from a Hashimoto patient. Subsequent studies showed that EBV-infected B lymphocytes from this patient synthesized IgG2 Tg antibody while unfractionated blood lymphocytes cultured with pokeweed mitogen secreted IgGI, IgG2, and IgG4 Tg antibodies in amounts proportional to those present in the patient's serum. To investigate this discrepancy further, we cultured EBV-infected lymphocytes from blood, lymph nodes, and thyroid tissue in medium alone and with increasing concentrations of PHA. In individuals with thyroid autoantibodies predominantly of subclass IgGI, PHA enhanced the levels of total Tg antibody synthesis without affecting the IgG subclass distribution. However, in patients with serum autoantibodies of subclasses IgGI, 2, and 4, the increased levels of total Tg antibody synthesis were associated with increased amounts of thyroid autoantibodies of all of these subclasses; in some instances IgGI and IgG4 autoantibodies were only synthesized in cultures containing PHA. These observations suggest that addition of the T-cell mitogen PHA to cultures of EBV-infected lymphocytes may ensure activation of B-cell precursors committed to synthesizing the IgG subclasses characteristic of serum antibody in the lymphocyte donor. Since Tg antibodies of subclasses IgG2 and IgG4 recog­ nize different epitopes on Tg, the ability to produce human monoclonal antibodies of different IgG sub­ classes may simultaneously ensure the development of antibodies to different epitopes on the same antigen.