PMC:2626671 / 5469-9124 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2626671","sourcedb":"PMC","sourceid":"2626671","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2626671","text":"Distinct expression kinetics of perforin and granzyme B during CTL development in culture\nOur 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-γ and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).\nFigure 1. Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway. Data are representative of at least five (A–E) or three (F) independent experiments.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).\nThese 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.","divisions":[{"label":"Title","span":{"begin":0,"end":89}},{"label":"Figure 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