Abstract
CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.
Discriminative SKP2 interactions with CDK-cyclin complexes support a cyclin A-specific role in p27KIP1 degradation
