A mixed lymphocyte culture system is described in which very small numbers of thymus or spleen cells from CBA (H-2) mice are cultured with allogeneic (H-2) cells, and the resultant cell-mediated lympholysis (CML) response of individual cultures is determined by Cr release from labeled target cells. Using this culture system and thymus and spleen cells from CBA mice of various ages, limit dilution analysis of CML responsiveness was carried out and estimates were thereby made of the frequency of alloreactive (anti-H-2) cytotoxic T lymphocyte precursors (CTL-P) in both organs during ontogeny. Fetal thymus cells contained no detectable CTL-P or cells capable of suppressing CTL-P responses from adult spleen cells in vitro. CTL-P were first detected in the thymus of 1-day-old mice. By 4 weeks of age their frequency had increased from an initial low value of 1/5 10 cells to 1/6.8; 10 cells, and there were 2,700 anti-H-2 CTL-P/thymus. The thymus of the 9-month-old mouse contained only 280 CTL-P at a frequency of 1/2 10 cells. CML responsiveness of the spleen was first detected at 3 days of age. The frequency of CTL-P in the spleen was initially low, but there followed a progressive accumulation of CTL-P until at least 9 months of age when the frequency was 1/3.5 10 cells. These results suggest a direct relationship between thymus and spleen CML responsiveness and that CTL-P initially generated in the thymus migrate and become CTL-P in the spleen. © 1979 by The Williams and Wilkins Co.
