Immune responses in vitro. 3. Development of primary gamma-M, gamma-G, and gamma-A plaque-forming cell responses in mouse spleen cell cultures stimulated with heterologous erythrocytes.

We have demonstrated for the first time that mouse spleen cells stimulated in vitro with heterologous erythrocytes developed immunoglobulin class-specific gammaM, gamma(1), gamma(2a+2b), and gammaA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse micro-chain antibody to the assay preparation, specifically prevented development of all gammaM PFC and enabled accurate and reproducible enumeration of immunoglobulin class-specific PFC after treatment with appropriate monospecific anti-globulins and complement. Culture conditions, with regard to medium, atmosphere, agitation, and spleen cell densities, were similar to those previously shown to support only gammaM PFC responses. Evaluation of the kinetics of appearance of PFC showed that gammaM PFC reached maximum numbers on days 4-5; the magnitude of this response was 3-10 times greater than gamma(1) gamma(2a+2b), or gammaA PFC which reached maximum numbers on days 5-6. Optimal erythrocyte antigen dose for gammaM PFC responses was 10(7)/culture, whereas a dose of 10(6) erythrocytes/culture consistently stimulated optimal gamma(1) gamma(2a+2b), or gammaA PFC responses. Investigations of the effects of anti-erythrocyte antibody on gammaM and gammaG PFC responses indicated that antibody suppressed these responses by neutralizing the effective antigenic stimulus at the macrophage-dependent phase of the response. At the same antibody concentration, gammaG PFC responses were more effectively suppressed than gammaM PFC responses. Further, gammaG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas gammaM PFC responses could only be completely suppressed during the first 24 hr. These results were discusssed in terms of the role of antigen in the stimulation gammaM and gammaG antibody.