text: Distinct expression kinetics of perforin and granzyme B during CTL development in culture
  Our experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1). Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A-D). Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A-D). In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E). As expected, a small fraction of naive T cells expressed the cytokines IFN-gamma and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A). 
  We evaluated antigen-dependent cytolytic function in a short-term assay in which target cell death was measured within 2 h (Fig. 1 F). By limiting the duration of TCR stimulation, this strategy minimizes cytolysis secondary to new gene expression during the period of the assay. Naive T cells did not display significant cytolytic function in this short-term assay (unpublished data), most likely because they express immature (unprocessed) forms of perforin and lack the capacity to degranulate (18, 19). Even after activation for 2 or 4 d, the cells showed poor cytolytic activity (Fig. 1 F), in striking contrast to their capacity for efficient cytokine production (Fig. 1 E). Only cells cultured until day 6 displayed robust cytotoxicity, as judged by their ability to induce apoptosis in a large number of target cells (Fig. 1 F). 
  These results show that after a strong priming stimulus through TCRs and co-stimulatory receptors in vitro, granzyme B expression and the ability to produce effector cytokines are programmed early, whereas perforin expression and cytolytic function are induced later, during the phase of clonal expansion in IL-2. Therefore, the two major effector functions of CTL, cytokine production and cytolytic activity, are not intrinsically coregulated. 
  