AUTORADIOGRAPHIC STUDIES ON THE IMMUNE RESPONSE

Abstract

The DNA-synthesizing capacity of single antibody-forming cells was tested by a combination of micromanipulatory and autoradiographic techniques. Rats were immunized with S. adelaide flagellin, a protein antigen known to contain significant contamination with somatic (O) antigen. Single cells from secondarily immunized rats were tested for production of anti-H and anti-O antibodies by previously described and newer techniques. Positive antibody producers were transferred onto clean dry slides by micromanipulation, and autoradiographs were performed. When rats had received tritiated thymidine 1 hour before killing, labeling of antibody-forming cells was taken to imply that the cell was preparing for further mitotic division. It was found that on the 2nd and 3rd day of a secondary response, many of the antibody-producing cells in the nodes (chiefly plasmablasts) were incorporating tritiated thymidine. At the height of the cellular response, however, at 4 and 5 days, the majority of active antibody producers (chiefly mature plasma cells) were incapable of DNA synthesis. There appeared to be an inverse relationship between the antibody-forming and DNA-synthesizing capacities of the cell population under study; as more of the cells studied formed detectable antibody, fewer of them incorporated the DNA precursor. The age of plasma cells was also studied. Animals were killed at the height of the cellular immune response, having previously received an injection of tritiated thymidine 1 to 48 hours before killing; i.e., at 63 to 110 hours after their secondary stimulus. As the interval between isotope injection and killing increased, the proportion of antibody-forming cells showing labeling increased. With an interval of 30 hours, about half the antibody-forming cells were labeled and of 48 hours, over 95 per cent were labeled. This was taken as evidence that, few, if any, antibody-forming cells found at the height of a secondary response were more than 48 hours old. On the basis of these experiments and those reported in the accompanying paper, a simplified scheme showing the development of an antibody-forming clone in the secondary response was proposed.