Serum factors affecting the incorporation of (3H)thymidine by lymphocytes stimulated by antigen. II. Evidence for a role of complement from studies with heated serum.

Lymph node cells from preimmunized rabbits were cultured with varying concentrations of antigen in autologous serum which had been collected before immunization. [H]Thymidine was added after 18 hours of culture and the cells were harvested at 24 or 66 hours for the determination of the radioactive labelling of acid-precipitable material. Preheating serum (56°, 20 minutes) enhanced labelling in both control and antigen-treated cultures. This `heat effect' had an early (24 hour) non-specific component, independent of antigen concentration, and a late (66 hour) specific component which was most evident at high antigen concentrations. The conditions of preheating serum (temperature and time) required to produce the heat effect were similar to those required to remove haemolytic activity against rat erythrocytes. However, at certain temperatures and times there were discrepancies. These discrepancies, and data from experiments in which preheated and unheated sera were mixed in varying proportions, or interchanged in different sequences, were explicable on the basis of (i) a requirement for complement in stoichiometric quantities dependent on the number of cells being inhibited, (ii) the involvement of the majority of the cultured cells in the early non-specific component of the heat effect, but only cells capable of proliferating in response to added antigen in the late specific component, (iii) the secretion of complement by cultured cells. Preheating serum (66°, 20 minutes) depressed labelling in control and antigen-treated cultures and reduced agglutinating activity against both autologous and heterologous erythrocytes. The results are discussed in relationship to models which require that the size of a specific lymphocyte clone be positively or negatively regulated by the concentration of antigen specific for that clone. With increasing antigen concentration three effects on cells bearing specific receptor sites are distinguished. (i) Cell stimulation under conditions of antigen concentration and cell receptor specificity such that only a few antigen molecules can bind to cells. (ii) Complement-dependent inhibition under conditions such that more antigen molecules can bind to cells. (iii) Complement-independent inhibition under conditions, possibly unphysiological, such that very large quantities of antigen molecules can bind to cells.