Fig. 3.

Inflammation regulates perforin and Dicer protein expression in differentiating CTLs. (A) Scheme of the in vitro differentiation system used to generate memory-like or effector CTLs from mouse P14 transgenic CD8⁺ T cells. (B) Perforin and Dicer expression measured by Western blot in activated CD8⁺ T cells 2 days after stimulation. Lane 1, lysates from CD8⁺ T cells stimulated for 2 days with anti-CD3 plus anti-CD28; lanes 2–4, lysates of CD8⁺ T cells isolated from splenocyte cultures 2 days after stimulation with gp33 ± CpG and IL-12 as indicated. (C) Perforin and Dicer expression measured by Western blot in CD8⁺ T cells activated with gp33 or gp33 plus CpG and differentiated for 6 days in the presence of high or low IL-2, with or without IL-12. Runx3 is used as a loading control. Results are representative of three independent experiments. (D) Real-time PCR analysis of Dicer mRNA levels in (Left) naïve and day 2 activated CD8⁺ T cells or (Right) day 6 CTLs derived from gp33-stimulated splenocytes and cultured with low or high IL-2, or IL-2 plus CpG and IL-12. (Mean +/− standard error.) (E) (Left) Sorting strategy of effector and memory CTL precursors based on the expression of CD127 and KLRG1, after gating on CD44^high CD8⁺ T cells, in LCMV-infected B6 mice. (Right) Western blot analysis of perforin and Dicer expression in naïve CD8⁺ T cells and in the four populations sorted according to KLRG1 and CD127 expression. Results are representative of two experiments with 10–14 mice each. Lanes marked with a red letter or number represent samples subsequently used for RNA sequencing.