Transient Appearance of Cells Secreting Antibody of Different Specificities after Immunization of Mice with Trinitrophenylated Erythrocytes

A sensitive variant of the method detecting hemolytic plaque-forming cells (PFC) was used to study the possibility of production of antibody of two different specificities by single cells. 2,4,6-Trinitrophenyl (TNP), conjugated pigeon erythrocytes (PRBC) were given in a single immunizing injection to mice, and their spleen cells were plated on days 3, 4, and 5 after this injection with both PRBC and TNP-sheep erythrocytes (SRBC). A significant number of PFC lysing both erythrocytes (3.24 × 106 cells) was found on the 3rd day after immunization. On the 4th day, out of 13.39 × 106 cells examined in all experiments, 6.260 (468 × 106 cells) monospecific PFC and 137 (10.2 × 106 spleen cells) bispecific PFC were found, whereas on the 5th day, only 0.74 × 106 cells were bispecific. Studies on cross-reactivity between PRBC and SRBC or TNP-SRBC showed that only 0.11 to 0.2 × 106 cells produce cross-reacting antibody. Furthermore, inhibition studies with TNP-bovine albumin or TNP-ε-aminocaproic acid showed that these inhibitors did not influence anti-pigeon PFC at all, but sharply reduced the number of anti-TNP-SRBC PFC and provoked a disappearance of bispecific PFC. On the other hand, short term incubation with puromycin also caused disappearance of bispecific PFC. The same proportion of double PFC was found after immunization of mice with native PRBC and SRBC. It can be inferred, therefore, that shortly (on the 3rd and 4th day) after primary immunization with two unrelated antigens, a low but significant percentage of cells simultaneously produce and secrete antibody of two different specificities.