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 γM, γ₁, γ_2a+2b, and γA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse µ-chain antibody to the assay preparation, specifically prevented development of all γM 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 γM PFC responses. Evaluation of the kinetics of appearance of PFC showed that γM PFC reached maximum numbers on days 4–5; the magnitude of this response was 3–10 times greater than γ₁ γ_2a+2b, or γA PFC which reached maximum numbers on days 5–6. Optimal erythrocyte antigen dose for γM PFC responses was 10⁷/culture, whereas a dose of 10⁶ erythrocytes/culture consistently stimulated optimal γ₁ γ_2a+2b, or γA PFC responses. Investigations of the effects of anti-erythrocyte antibody on γM and γG 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, γG PFC responses were more effectively suppressed than γM PFC responses. Further, γG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas γM 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 γM and γG antibody.