The maintenance of lytic specificity during the development of clones of cytotoxic T lymphocytes from single precursor cells.

A high-cloning efficiency, filler cell-free limit-dilution culture system for the growth and differentiation of single cytotoxic T lymphocyte precursors (CTLp) was tested for its ability to maintain the lytic specificity of the resultant clones of cytotoxic T lymphocytes (CTL). The system used non-specific stimulation with phorbyl ester and calcium ionophore, maintenance of growth over the first 6 days of culture with interleukin (IL)-2 and interferon-gamma, and maintenance of growth and differentiation over the last 2 days of culture with IL-2 and IL-6. Under these defined conditions around 50% of all CD4- 8+ T cells developed into CTL clones that were specific in their lytic activity. In contrast, a culture system maintained by irradiated filler cells showed non-specific lysis of both YAC-1 type natural killer targets and of P815 type targets, while a culture system maintained by IL-2 and a crude growth factor preparation showed non-specific lysis of natural killer targets but not of P815. The defined lymphokine culture system was suitable for determining the specificity repertoire of primary CTLp. Using this system, the frequency of reactivity with allogenic tumor targets was found to be approximately one CTLp in 30 for several mouse strain/target cell combinations.