FIGURE 8.

Inhibition of ERK signaling promotes differentiation of naive peripheral αβ CD8⁺ T cells into cells with memory-like characteristics. (A) Differentiation of naive WT F5 Rag2^−/− CD8⁺ T cells. Peripheral CD8⁺ T cells were activated for 2 d with anti-CD3/anti-CD28 (10 μg/ml), with or without U0126 addition, and then cultured in IL-2 as indicated. Intracellular activated phospho-ERK (top) or Egr-1 protein (bottom) following anti-CD3 and anti-CD28 activation. Gray shading, isotype control; color-coded lines, WT F5 Rag2^−/− cells according to U0126 concentration. (B) Expression of Eomes and T-bet. Top, Quantitative RT-PCR analysis. Bottom, Flow cytometric analysis of protein levels. (C) Flow cytometric analysis of intracellular IFN-γ and granzyme B expression after PDBu/ionomycin activation for 5 h. Data are representative of six (A–C) independent experiments, each done in triplicate. Data are mean ± SEM. Statistical significance: ***p < 0.001 (unpaired t test). (D) Summary of the relationship between MHC-peptide–TCR avidity, activation of TCF-SRF target genes, and cell differentiation pathways in thymus and periphery. Inactivation of ERK-ELK4/ELK1–SRF signaling in thymocytes and in peripheral αβ CD8⁺ T cells favors the development of cells with innate-like characteristics, which exhibit enhanced Eomes expression and immediate production of IFN-γ.