The in vitro generation of antigen-binding cells

Abstract

To analyze early events following the contact of antigen with specific receptor-bearing cells, antigen-binding cells (ABC) specific for sheep erythrocytes (SRC) were generated in a primary Mishell-Dutton culture system. The maximal frequency of ABC (a five-fold increase over Day 0 involving both T and B cells) occurred on Day 4 in culture conditions which yielded optimal numbers of plaque-forming cells (PFC) specific for SRC 1 day later. Assessment of the total Ig-secreting cells as well as ABC and PFC for ox and burro erythrocytes showed that the ABC as well as the PFC response to SRC was antigen specific. By autoradiography of [ 3H]thymidine-pulsed cultures and by inhibiting cell division with hydroxyurea, the continuous contribution of cell division to the generation of ABC was demonstrated. As with PFC, the decline in ABC numbers began at the point where their generation by cell division slowed. After removal of hydroxyurea, ABC frequency and thymidine incorporation recovered substantially, but PFC did not. However, division of ABC was not the only process involved in ABC generation. Indeed, 20% of the Day 4 ABC arose from cells dividing in the first 24 hr of culture which were not detectable as ABC at that time, i.e., some ABC were derived from dividing non-ABC. Thus both cell division and maturation contributed to the increase in specific ABC seen after primary in vitro immunization. Both processes can be added to the list of early antigen-stimulated events in specific ABC which can be analyzed in the primary culture system in future studies of cell interactions.