CD4⁺CD25⁺ regulatory T-cell lines from human cord blood have functional and molecular properties of T-cell anergy

CD4⁺CD25⁺ regulatory T cells (Tregs) are essential negative regulators of immune responses. Here, we examined the signaling properties of human Tregs, using CD4⁺CD25⁺ Treg and CD4⁺CD25⁺ control (Tcont) cell lines generated from cord blood. Treg cell lines were markedly hyporesponsive to stimulation with dendritic cells and with anti-CD3/CD28-coated beads. Hyporesponsiveness was reversed by exogenous interleukin-2 (IL-2). T-cell receptor (TCR)-CD3/CD28- mediated activation of Rap1 and Akt was retained in Tregs, but activation of Ras, mitogenactivated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase 1/2 (Erk1/2) was impaired. Tregs were blocked from cell cycle progression due to decrease of cyclin E and cyclin A and increase of p27^kip1 (p27^kip cyclin dependent kinase inhibitor). IL-2 induced sustained increase of cyclin E and cyclin A and prevented up-regulation of p27^kip1. Tregs had high susceptibility to apoptosis that was reversed by IL-2, which correlated with activation of Erk1/2, up-regulation of Bcl-X_L (B-cell CLL/lymphoma 2-like nuclear gene encoding mitochondrial protein, transcript variant 2), and phosphorylation of Bad (Bcl2 antagonist of cell death) at Ser¹¹². Thus, Tregs share biochemical characteristics of anergy, including abortive activation of Ras-MEK-Erk, increased activation of Rap1, and increased expression of p27^kip1. In addition, our results indicate that TCR-CD3/CD28-mediated and IL-2 receptor-mediated signals converge at the level of MEK-Erk kinases to regulate Treg survival and expansion and suggest that manipulation of the MEK-Erk axis may represent a novel strategy for Treg expansion for immunotherapy.