In vitro stimulation of antibody formation by peritoneal cells. 3. Effect of active immunization on the subsequent in vitro performance of peritoneal and spleen cells.

Male CBA mice were given a single intraperitoneal injection of sheep red blood cells (SRBC) or horse red blood cells (HRBC). They were killed at intervals of 1–10 days thereafter, and micro-cultures of spleen cells or peritoneal cells (PC) were prepared. These consisted of a thin film of tissue culture medium containing carboxymethyl cellulose (CMC), mouse lymphoid cells, guinea-pig complement and either SRBC or HRBC, held at 37° under liquid paraffin. Cultures were read repeatedly for appearance of haemolytic plaques. PC from SRBC-immunized mice showed an altered reactivity on SRBC monolayer cultures. The peak plaque count achieved fell progressively for 4 days after immunization, and then returned to normal by day 7. The actinomycin D resistant component of the PC response rose rapidly; at 1 day after immunization it was equal to the total response. Over the next 3 days after immunization it fell again to normal levels. The results suggested that the injection sets in train events locally in the peritoneal cavity which resembled those following culture of normal PC in SRBC monolayers. The effects were immunologically specific as only marginal changes followed the injection of HRBC. Spleen cells from SRBC-immunized mice, when cultured in SRBC monolayers, yielded many cells capable of giving plaques after 5–60 minutes incubation, as expected. These were deemed to be cells forming antibody at the moment of killing of the animal. In addition, such cultures developed new plaques over the subsequent 23 hours in culture. These were produced by cells not initially forming antibody which switched into antibody secretion at some time during culture. At early time points after immunization, this second type of cell was much more numerous than the first type. The switch from non-secretor status could occur in the presence of a high concentration of actinomycin D. Operationally these non-secretors in immunized spleens resembled an important fraction of PC from unimmunized retired breeder mice. The progressive conversion of non-secretor cells into secretors, if it occurs , would have a major influence on the kinetics of appearance of PFC in a spleen after immunization. While spleen cells from mice immunized with HRBC performed on HRBC monolayers much as described above, PC from HRBC-immunized mice could not be induced to cause significant lysis in HRBC monolayers. The same was true of PC from mice chronically fed with HRBC. In fact, no method has yet been found to persuade PC to produce lytic plaques active against erythrocytes other than SRBC.