Polyreactive antibodies (Ab) are naturally occurring Abs, primarily of the IgM type, and are capable of reacting with a wide variety of antigens (Ags). As the biological function of polyreactive Abs, polyreactive Ab- producing B cells, and the molecular structure of the paratope responsible for multiple Ag binding are still unclear, the objectives of this study are: 1) to find a cellular marker which can be used to identify human B cells making polyreactive Abs; 2) to study the function(s) of polyreactive Ab-producing cells; and 3) to define molecularly the paratope structure responsible for multiple Ag-binding of polyreactive Ab. Ag-binding and non-Ag-binding B cells were isolated by FACStarplus and analyzed for their ability to make polyreactive Abs. Four to six times more cells making polyreactive Abs were found in the B cell subset that bound Ags than in the B cell subset that did not bind Ags. FACS analysis revealed that cell lines making polyreactive Abs bound a variety of Ags, whereas cell lines making monoreactive Abs bound only a single Ag. Both CD5+ and CD5- Ag-binding B cells made polyreactive Abs, but the frequency was slightly higher in the CD5+ Ag-binding (85%) as compared to the CD5- Ag-binding (50%) population. Comparison of Ag-binding and non-Ag-binding CD5+ B cells showed that approximately 86% of the former, but only 15% of the latter, made polyreactive Abs. FACS analysis further revealed that few Ag-binding B cells express B7 molecules (i.e. B7-1 and B7-2). Examination of the frequency of the polyreactive Ab-producing precursor cells in the B7+/non-Ag-binding and the B7-/Ag-binding B cell populations showed that approximately ten times more polyreactive Ab-producing precursor cells were found in the latter than in the former. No upregulation of B7-1 and/or B7-2 expressions were observed on B cells up to 48 hrs after incubation with thyroglobulin (Tg) and beta-gal which these cells bound with low affinity. In contrast, a strong upregulation of B7-1 and B7-2 expression were detected after 24 hrs incubation with anti-human Ig xenoantibodies which B cells bound with high affinity. In other experiments 125I-Ag was incubated with a human polyreactive Ab producing hybridoma cell line (mAb63), and the conversion of TCA-insoluble 125I-Ag protein into TCA-soluble 125I-Ag peptides was determined. We found that the polyreactive cell line was able to process 125I-IgGFc, -Tg and - insulin, although not as efficiently as it processed 125I-anti-human Ig xenoantibodies which bound with high affinity. In contrast to polyreactive hybridomas, monoreactive hybridomas processed only the Ag to which the surface Ab reacted. Our results suggest: 1) Ag-binding can be used as a marker to identify polyreactive Ab-producing precursor B cells; 2) polyreactive Ab-producing precursor cells are B7 negative cells; 3) low affinity Ag binding does not lead to the upregulation of B7 molecules; and 4) polyreactive Ab-producing cells can process multiple Ags. The study on the molecular structure of the polyreactive Ab paratope was only recently initiated and is progressing.