Bispecific cells among IgM and IgG producers during the early phase of primary and secondary responses

Simultaneous immunization of mice with sheep (SRBC) and horse (HRBC) erythrocytes regularly resulted in the appearance of hemolytic plaque‐forming cells (PFC) specific for each type of erythrocyte and also of PFC lysing both types of erythrocytes. After primary stimulation the highest number of bispecific cells (42/106 cells) was found among PFC as revealed by the direct procedure (IgM producers). Among PFC enhanced with anti‐mouse Fab serum (IgG producers), bispecific cells were less numerous (8/106 cells). In preparations enhanced by anti‐mouse‐γFc serum which reveals IgG producers without inhibiting IgM antibody, the number of bispecific PFC equalled the sum of bispecific cells revealed by direct and anti‐Fab enhanced procedures. The number of direct bispecific PFC during primary and secondary response was approximately the same, whereas the number of IgG‐producing, bispecific PFC increased considerably during the secondary response. Another difference was the time limitation of the appearance of bispecific cells: after primary immunization direct bispecific PFC were detected only on days 3, 4 and 5, but enhanced bispecific PFC were present from day 4 up to day 12. However, during the secondary reaction, bispecific PFC were detected by all three procedures only between days 3 and 6. Studies on the cross‐reactivity between SRBC and HRBC gave negative results at the humoral levels, even when the mice were primed with a minimal amount of both erythrocytes and then two months later, boosted with one of them. Studies at the cellular level showed that after immunization with one antigen, only 0.4 to 0.7 direct or enhanced PFC/106 cells could simultaneously lyse both erythrocyte types. Thus, a hundred times more bispecific PFC were constantly found after double immunization of the animals. Moreover, sudden disappearance of all bispecific PFC on the 7th day after secondary stimulation makes it unlikely that all bispecific PFC are simply cross‐reacting cells.