In Vitro Proliferation of T Lymphocytes from Listeria -Infected Rodents: Assay Conditions for Rat Peritoneal Exudate Cells and Characterization of an Inhibitor

Conditions favorable to [(3)H]thymidine incorporation into antigen-stimulated T lymphocytes from Listeria-infected rats have been established. In cultures of peritoneal exudate (T) lymphocytes purified twice with nylon-wool vigorous antigen-specific proliferation was observed within 2 days. Cultures of lymphocytes from nodes draining a subcutaneous Listeria-infection site differed in that back-ground proliferation was higher than for peritoneal exudate lymphocytes, and [(3)H]thymidine incorporation was maximal at day 3. A critical factor for the rate of proliferation was the lymphocyte-to-macrophage ratio; optimal cultures of peritoneal exudate lymphocytes contained 2 to 5% macrophages. Macrophages exceeding a proportion of 10% strongly, if not completely, inhibited [(3)H]thymidine incorporation into antigen-stimulated lymphocytes. Inhibition was associated with mononuclear cells, adherent to plastic or nylon-wool, of the stimulated or unstimulated peritoneal cavity. It was neither attributable to release of cold thymidine from macrophages nor to rapid degradation of particulate antigen by macrophages. The degree of inhibition reflected the metabolic activity of macrophages; on a cell-for-cell basis, heat-killed and glutaraldehyde-fixed macrophages were less inhibitory, and stimulated macrophages were more inhibitory than macrophages from the unstimulated peritoneal cavity.