Article Figures & Data
Figures
- FIGURE 1.
IL-2 promotes survival of IL-2-dependent T cells via the PI3K/PKB pathway. A, IL-2-dependent CTLL-2 mouse T cells were grown in the absence of IL-2. After 24 and 48 h they were harvested, fixed in ethanol, and labeled with propidium iodide to determine their DNA profiles by flow cytometry. B, CTLL-2 were deprived of IL-2 for different periods of time and harvested. They were monitored for PKB expression by Western blotting using a rabbit anti-phospho-Ser473-PKB Ab, recognizing active PKB (upper panel), or an Ab directed against total PKB (lower panel).
- FIGURE 2.
Specific interference with the PI3K/PKB pathway mimics IL-2 deprivation. A, CTLL-2 mouse T cells were treated with LY294002 for 24 and 48 h in the presence of IL-2. They were harvested, fixed, and labeled as described in Fig. 1A for FACS analysis. B, CTLL-2 were treated with LY294002 for the corresponding periods of time in presence of IL-2, harvested, and lysed. PKB activity and total PKB were determined using the same Abs as in Fig. 1B.
- FIGURE 3.
IL-2 signaling regulates forkhead transcription factor activity via the PI3K/PKB pathway. A, The expression pattern of different FoxO forkhead transcription factors was determined in CTLL-2 mouse T cells. For this purpose, CTLL-2 cells were lysed and analyzed by Western blotting using an anti-phospho-Thr24-FoxO1a/phospho-Thr32-FoxO3 (/phospho-Thr28-FoxO4) Ab (upper panel) or an anti-total FoxO4 Ab (lower panel). Controls consisting of FoxO4-, FoxO1-, and FoxO3-transfected U2OS cells were loaded in parallel. B, CTLL-2 were deprived of IL-2, lysed after different periods of time, and analyzed by Western blotting using a rabbit anti-Thr32-phospho-FoxO3 Ab (upper panel) or an Ab recognizing total FoxO3 (lower panel). C, CTLL-2 were treated for different periods of time with LY294002, lysed, and analyzed by Western blotting using the same Abs as in B.
- FIGURE 4.
FoxO3 directly regulates p27Kip1 and Bim expression. A, CTLL-2 were either deprived of IL-2 (upper panels) or treated with LY294002 (lower panels) for different periods of time, harvested, and lysed. p27Kip1, Bim, and actin protein levels were monitored by Western blotting using the appropriate Abs. B, CTLL-2 cells were deprived of IL-2 for different periods of time and lysed. Using an anti-Fas ligand Ab, the expression levels of this protein were analyzed by Western blotting to determine whether forkhead transcription factors induce other important proapoptotic target genes in these cells. C, CTLL-2 were deprived of IL-2 for the time indicated and treated or not with actinomycin D, a transcription inhibitor. RNAs were extracted at the indicated time points after IL-2 withdrawal and analyzed by Northern blotting using a p27Kip1 cDNA probe and a GAPDH cDNA probe as loading control.
- FIGURE 5.
PKB-independent activation of FoxO3 mimics IL-2 withdrawal. A, CTLL-2 cells were electroporated with FoxO3(A3)ER construct. Clonal cell lines expressing this construct in a stable manner were selected on G418. The clones were harvested, lysed, and analyzed by Western blotting using an anti-hemagglutinin mAb. Clones 2 and 6 appeared to express similar levels of FoxO3(A3)ER. B, Clones 2 and 6 were treated with 4OH-T for 24 h in presence of IL-2 and analyzed by FACS as described in Fig. 1A. C, Clone 6 was treated with 4OH-T in the presence of IL-2 for different time lapses and analyzed by Western blotting using the appropriate Abs. D, Clone 6 was treated with 4OH-T in presence of IL-2 for the reported time lapses, harvested, and lysed. RNAs were isolated and equal amounts were analyzed by Northern blotting as described in Fig. 4C.
- FIGURE 6.
Activation of peripheral T lymphocytes is linked with PI3K/PKB-dependent phosphorylation of FoxO1. A, Primary T cells were isolated from fresh blood. The cells were stimulated with anti-CD3 mAbs and IL-2 in the presence or absence of LY294002. The cells were harvested after 24 and 48 h, fixed, and stained for FACS analysis as described in Fig. 1A. B, Protein samples prepared from the cultures shown in A were separated on polyacrylamide gels, and FoxO1a-Thr24P levels were detected on Western blot. C, Primary T cells were isolated from fresh human blood and stimulated on UCHT-1 anti-CD3-coated dishes for different periods of time. The CD25+ cell population was determined by FACS analysis by incubation with anti-CD25-PE Abs. D, Primary T cells were stimulated with anti-CD3 Abs for 24 h, and then with IL-2 for the indicated times. Cells were harvested, lysed, and analyzed by Western blotting with an anti-phospho-Thr24-FoxO1a Ab.
- FIGURE 7.
The IL-2 downstream pathway bifurcates at the level of the forkhead transcription factors to modulate both cell cycle and apoptosis. Early in an immune response, IL-2 stimulation promotes proliferation of newly Ag-primed CD25+ T lymphocytes through activation of the PI3K/PKB pathway. Activated PKB phosphorylates FoxO members of the forkhead transcription factor family, thereby preventing their translocation to the nucleus and thus transcription of the cell cycle inhibitor p27Kip1, allowing the T cells to proliferate. Later in the immune response, when Ag and IL-2 become limiting, withdrawal of IL-2 shuts down the PI3K/PKB pathway, releasing active FoxO forkhead transcription factors which can in turn activate transcription of target genes, such as p27Kip1 and Bim. The Cdk inhibitor p27Kip1, via its brake activity on the cell cycle progression, induces an arrest in G1 phase. Bim, a proapoptotic member of the Bcl-2 family, can induce apoptosis in the activated T cell pool. In conclusion, the IL-2 signaling pathway regulates the FoxO members of the forkhead transcription factor family and bifurcates at that level to exert a dual effect on both cell cycle and cell death via p27Kip1 and Bim, respectively.
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