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The clonal nature of antibody formation. I. Clones of antibody-forming cells of poly-D-alanine specificity.

With the aid of a cell transfer system using F 1 -hybrid spleen cells of syngeneic mice, poly-d-alanine (poly-ala) antigen and a modified antibody plaque assay, we were able to rigorously test and confirm the clonal nature of antibody formation. Our results showed that when x-rayed mice were repopulated with relatively small numbers of spleen cells followed by two immunizations, only a few animals produced antibodies to poly-ala and in the spleens of most of these responder mice only one of two allelic gene regions determining 7S antibody phenotype (B or C allotype) was expressed. On the other hand, when relatively large numbers of spleen cells were transferred to x-rayed mice followed by two immunizations, all recipients responded to poly-ala and expressed both antibody-forming cells of B and C allotype. The all-or-none response characteristics at limiting spleen cell doses were amenable to Poisson statistics suggesting that some mice were repopulated by descendants of a single poly-ala-sensitive precursor unit, i.e., by an individual clone of antibody-forming cells. The application of Poisson statistics to mice expressing only one marker allotype confirmed this interpretation. The number of mice expected to be repopulated by a single poly-ala-sensitive precursor unit correlated very well with the number of responder mice expressing only one allotype. Thus antibody specificity and allotype must be transmitted together in cell lines and thereby characterize individual clones. The total number of prospective clones of poly-ala specificity was estimated to be about 150 per young adult mouse. The calculated size of a given individual clone was 20,000 to 50,000 synthesizing cells which represented about 14 to 16 cell divisions.