The analysis of an anti-fluorescein cytotoxic response

The detailed kinetics of the appearance of clones of anti-fluorescein cytotoxic T lymphocytes (CTL) in a primary limiting dilution response have been examined. The addition of factors from the supernatants of concanavalin A-stimulated rat spleen cell cultures was necessary to obtain linear dose-response curves and strong primary responses. In a standard primary response the maximum detectable number of specific clones was found, on day 5, to be derived from precursors having a frequency of 1 in 10 000 spleen cells. When limiting dilution cultures were restimulated on day 3, responses on day 5 were unaffected but new clones appeared on day 7 with a frequency of 1 in 1000. The emergence of the day-7 response depends on the addition of factor(s) on day 3. It is proposed that the early low frequency precursors detected on day 5 and the high frequency precursors detected on days 7-9 are derived from two stages in the differentiation lineage of CTL precursors. Restimulation of limiting dilution cultures involves not only the continued clonal expansion of primary responses but also the emergence of new "clones" of specific CTL.