Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.

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