GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO III. Velocity Sedimentation Studies of the Differentiation and Fate of Effector Cells in Long-Term Mixed Leukocyte Cultures* BY H. ROBSON MAcDONALD,$ JEAN-CHARLES CEROTTINI, AND K. THEODOR BRUNNER

The cell-mediated immune response to primary allogeneic stimulation is characterized by the appearance of cytotoxic T lymphocytes (CTL) 1 (see reference 1 for review). Recently, the existence of anamnestic CTL responses to allografts has been suggested by studies in which the ability of normal and alloimmune spleen cells to respond to relevant alloantigens both in vivo (2, 3) and in vitro (4) was quantitatively compared. In other studies, physical characterization of CTL in the spleens of mice undergoing tumor allograft rejection revealed significant differences in size and density between effector cells detected early or late after immunization (5-9), the former being predominantly large and of low density, whereas the latter were mainly small and dense. In view of these observations, two important questions regarding the differentiation and ultimate fate of CTL can be raised: (a) Do the changes in physical properties of CTL with time reflect the differentiation of a single cell lineage, or alternatively the parallel development of several cell lineages? (b) Are the cells capable of mounting an anamnestic response to alloantigens derived from CTL, or are CTL truly end cells capable of no further differentiation? Until individual CTL can be identified and isolated, no definitive answer to these questions is possible. However, with the concomitant development of in vitro systems for the generation of CTL (10-12) and quantitative assays for the estimation of their relative frequency (1, 12), indirect approaches to the study of the differentiation and fate of CTL are now feasible. In a previous paper of this series (13), we presented evidence that an anamnestic response to alloantigens could be demonstrated in long-term mixed leukocyte cultures (MLC). In this