Studies on cytotoxic lymphocyte production in culture

Abstract

Clones of cytotoxic T lymphocytes (CTL) responding to the hapten fluorescein have been examined by limiting dilution analysis. The analysis was carried out to determine the frequency of anti-fluorescein CTL precursor cells, the size of clones of cytotoxic cells, and the specificity of the cytotoxicity generated. Two limiting dilution culture procedures were developed to examine both the appearance of clones of anti-fluorescein CTL with time, and the stages in the CTL developmental pathway. 1. Limiting dilution cultures of normal spleen cells that received growth factors and stimulator cells at the start of the culture period. The precursor cell frequency reached a maximum value on days 4 to 6 at 1 precursor in 8-10,000 spleen cells in this system. 2. Limiting dilution cultures of normal spleen cells that received growth factors and stimulator cells on both Days 0 and 3 of the culture period. The precursor cell frequency reached a maximum value of 1 in 1000 spleen cells later in the response on Days 7 to 9 in this restimulated system. The precursor cells giving rise to clones of CTL on Day 5, and those giving rise to clones of CTL on Day 7 were interpreted as belonging to two subpopulations of precursor cells. The possibility that these two subpopulations represented separate stages of the anti-fluorescein CTL developmental pathway was suggested. The specificity of the cytotoxicity generated in restimulated cultures was dependent on both the source and quantity of growth factors. When high concentrations of untreated growth factors obtained from rat spleen cell cultures were added, non-specific cytotoxicity was generated. When the growth factors were semi-purified by size-column and ion-exchange column chromatography, highly specific anti-fluorescein cytotoxicity was generated. Non-specific growth factors other than Interleukin 2 were found to be required for the production of anti-fluorescein CTL.