Requirements for growth of immature thymocytes from fetal and adult mice in vitro.

We report here defined culture conditions that allow reproducibly the growth of the majority of immature thymocytes from both fetal (14-15 days of gestation) and adult mice. The combination of phorbol myristate acetate (PMA), ionomycin and recombinant interleukin 2 (IL2) is both sufficient and necessary to induce growth of about 1/6.2 (range 1/3-1/9) and 1/4.3 (range 1/2-1/7) immature thymocytes from adult and fetal mice, respectively, in serum-free cultures. Several other combinations tested (e.g. PMA + IL2, concanavalin A + IL2) were poorly or not active. None of the agents tested alone (PMA, ionomycin, concanavalin A, pokeweed mitogen, IL2) had any effect. We found no evidence for a role of IL1 and IL3 on growth of these cells. The growth of activated immature thymocytes from either fetal or adult mice was inhibited by a monoclonal antibody against mouse IL2 receptors. Under the same conditions that stimulated growth of most immature thymocytes, they did not mature into cells expressing Lyt-2, L3T4 or T cell antigen receptor (KJ16) after 7 to 15 days of continuous proliferation in culture. Nor did they give rise to cells with cytolytic activity after 7-9 days of culture. In some but not all experiments cultures of immature thymocytes from adult mice but not from fetal mice generated cells (1 out of 120-310) with helper function for B lymphocytes. While we confirmed here that approximately 50-70% freshly isolated immature thymocytes express receptors for IL2, our results indicate that these cells need to be activated (by e.g. PMA + ionomycin) to respond to IL2. A possible mechanism to account for the expression of nonfunctionally competent IL2 receptors is proposed and our results concerning the maturation of immature thymocytes in vitro are discussed.