A. Studies of the effects of IL-12 on CTL differentiation were pursued. IL-12 was found to have significant dose promoting and differentiating effects and to have complex interactions with IL-2 and IL-4 which are highly dose- and timing-dependent. B. Studies of human CTL lines developed and grown in the presence of IL-4 rather than IL-2 have continued. Their differentiation into the cytotoxic phenotype are now under study. C. Techniques have been established for the PCR based measurement of human mRNA for perforin and granzyme A - two protein constituents of cytolytic lymphocyte granules. The sensitivity of this technique will allow use in early clones as well as bulk culture. D. The high efficiency cloning system for human CTL precursors developed previously was employed for limiting dilution analysis of CTL differentiation. Clones of activated lymphocytes are being produced and studied for the effects of cytokines on expression of genes involved in the cytotoxic phenotype, i.e. perforin and granzyme A.
