Culture conditions for induction of suppressor cells in vitro

Culture conditions have been established for optimal generation of cytotoxic T lymphocytes (CTL) in one-way mixed lymphocyte cultures (MLC). The responder cell concentration was found to be most critical. In general the more active the proliferation was, the lower was the optimum for CTL generation. Optimal proliferation was usually found to occur at lower cell concentrations than optimal CTL generation. Above the optimal CTL generation proliferation also decreased, most likely due to overcrowding. These observations were made on both unpurified and nylon wool-passaged spleen cells, though the latter generally produced higher levels of cytotoxic activity per equal number of harvested cells. Cells prestimulated for 2 or 4 days in the absence or presence of antigen (M-locus differences) were unable to mount an effective response when restimulated with another antigen presenting an H-2 difference. This hyporesponsiveness was not due to the exhaustion of the culture medium or the presence of dead cells (since these were removed before restimulation). A high degree of hyporesponsiveness was also induced in the absence of antigen during prestimulation which rendered antigenic competition highly unlikely. Overcrowding could be excluded since the observed inhibition could not be diluted out by lowering the responder cell concentrations. A carry-over of CTL destroying newly added antigen (Fitch et al., 1975) could not account for our observations since in the system utilized (M-locus stimulation) there was no generation of CTL (Festerstein, 1973; Röllinghoff et al., 1975). The development of suppressor cells would seem the most likley explanation of the data.