Some limits to post-antigen generation of diversity: failure to detect variants in clones of hapten-specific antibody-forming cells (AFC) developing in culture from direct AFC-progenitor B cells.

A search was made for variants in clones of hapten-specific antibody-forming cells (AFC) arising by stimulation of mature B cells with either thymus-independent hapten-POL (polymerized bacterial flagellin) conjugates, or the polyclonal activator lipopolysaccharides. Enriched, hapten-binding B cells or unfractionated spleen cells were cultivated for 3-4 days at limiting dilution in the presence of thymus filler cells, and the AFC in each microculture well were then assayed for plaque formation on various hapten-sheep red cell monolayers. No variants were found from (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) to 2,4-dinitrophenyl specificity, nor from fluorescein (FLU) to NIP specificity. No variants were found in avidity for FLU hapten. All 374 clones examined, including clones of up to 300 AFC, appeared to be homogeneous in antibody specificity and plaque morphology under our conditions. These results differ from published findings using erythrocytes as antigens. Reasons for this discrepancy are discussed, including differences in sensitivity differences in immunological similarity between the test antigens, and in the particular B cell subsets involved.