Induction of haemolytic plaque-forming cells with sheep lymphoid cells in vitro

The in vitro induction of specific primary and secondary immune responses in sheep lymph node cell suspensions is described and suitable culture conditions determined. The induction of primary immune responses required supplementation of the culture medium with antigen-absorbed homologous serum or lymph, whereas the requirements for the induction of a secondary response were less stringent. The addition of 2-mercaptoethanol to the medium was required. The amounts of heterologous erythrocytes used for immunization were critical and optimal responses were obtained when 50 microleters of a 1% suspension were added to 1 ml cultures. Lymphocyte densities of about 5 X 10(6)/ml were found optimal in primary immune responses in vitro. Less than 2 X 10(6) cells/ml rarely gave rise to plaque-forming cell (PFC) generation, whereas densities of 10 x 10(6) and above reduced the number of PFC obtained per number of cultured cells. Lymphocytes obtained from the efferent lymph draining lymph nodes previously immunized with heterologous erythrocytes were found to generate PFC in vitro when specific antigen was added to the cultures, but attempts to generate PFC in vitro with cells from efferent lymph draining non-immunized nodes failed.