Article Figures & Data
Figures
- Figure 1.
The inhibition of PI3K and Akt in human T cells selectively inhibits the proliferation of human Tregs compared with Tconvs in a dose-dependent manner. Tregs (CD4+CD25HI) and Tconvs (CD4+CD25−) were fractionated from human PBMCs by cell sorting. Proliferation was examined by dilution of CFSE after stimulation with anti-CD3/anti-CD28 for 3 days with titrated amounts of triciribine (TCN), MK-2206, wortmannin (WM), and IC87114. Dead cells were excluded by 7-AAD incorporation. Left, average of three experiments, normalized to untreated controls; right, representative examples. A, a significant reduction in Treg proliferation was observed in response to Akt inhibition by triciribine compared with that of Tconvs at all doses tested: 5 μmol/L (P = 0.04), 10 μmol/L (P = 0.03), and 50 μmol/L (P = 0.01). B, a significant reduction of Treg proliferation in response to MK-2206 treatment was observed compared with that of Tconvs at the 1 μmol/L (P = 0.05) and 5 μmol/L (P = 0.005) doses. C, proliferation of Tregs was significantly reduced by wortmannin compared with that of Tconvs at all doses tested: 300 nmol/L (P = 0.03), 800 nmol/L (P = 0.01), and 1,000 nmol/L (P = 0.002). D, Treg proliferation was significantly reduced by IC87114 compared with that of Tconvs at the 10 μmol/L (P = 0.04) and 20 μmol/L (P < 0.005) doses. *, P < 0.05.
- Figure 2.
PI3K and Akt inhibition differentially inhibits the proliferation and downstream activation of murine Tregs compared with that of Tconvs. Mouse CD4+ splenocytes were fractionated into CD25+ (Treg) and CD25− (Tconv) subsets using magnetic bead enrichment kits. Proliferation was assayed by the dilution of CFSE after stimulation with anti-CD3/anti-CD28 for 3 days with wortmannin (WM; 200 nmol/L), MK-2206 (2 μmol/L), IC87114 (10 μmol/L), or DMSO. Dead cells were excluded by 7-AAD incorporation. A, representative example of the selective proliferation inhibition in Tregs (bottom) by wortmannin, MK-2206, and IC87114 compared with that of Tconvs (top). B, treatment with wortmannin, triciribine (TCN), IC87114, or MK-2206 resulted in a marked decrease of phosphorylated S6 in Tregs (CD25+) in comparison with Tconvs (CD25−). C, densitometry of phosphorylated S6 levels to S6 ratio showed significantly lower levels in Tregs when compared with Tconvs when the stimulated cells were treated with PI3K–Akt pathway inhibitors. Densitometry was performed on gel as shown in B.
- Figure 3.
The PI3K–Akt pathway is essential for the in vivo maintenance of murine Tregs. Mice were conditioned with wortmannin (WM; 40 μg), triciribine (TCN; 50 μg), MK-2206 (10 μg), or DMSO for 1 week on alternate days before flow cytometric analysis of splenocytes. Alternatively, mice were conditioned on alternate days with either wortmannin or DMSO for a week before vaccination with E7. One week after E7 vaccination, splenocytes were harvested, and the E7 immune response was assayed by ELISPOT. A, mice treated with DMSO vehicle contained similar percentages of CD4 T cells compared with wortmannin-, triciribine-, or MK-2206–treated animals (P = 0.1). B, the number of FoxP3+ cells in CD4+ T cells was significantly reduced in the mice treated with the inhibitors compared with DMSO (P < 0.05). C, vaccination resulted in a significant increase in E7-reactive T cells. Wortmannin and triciribine treatment significantly increased the number of E7-reactive cells compared with DMSO-treated controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 4.
PI3K–Akt signaling inhibition mitigates tumor growth. A, TC-1 tumor-bearing mice were treated with wortmannin (WM) or DMSO for 1 week after palpable tumors were detected. Mice were then vaccinated with E7 peptide and tumor growth was monitored. Both E7 vaccination (P < 0.05) and wortmannin treatment (P < 0.001) significantly inhibited TC-1 tumor growth, yet the greatest impairment was achieved with the combination of wortmannin treatment and E7 vaccination (P < 0.0001). B, mice were prophylactically treated with wortmannin, triciribine (TCN), or DMSO before s.c. B16 tumor inoculation and tumor growth was monitored. Tumor growth was significantly inhibited by wortmannin and triciribine compared with DMSO-treated animals (P < 0.0001). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 5.
Akt inhibition by MK-2206 enhances the antitumor therapeutic effect of tumor-specific vaccine. TC-1 tumor-bearing mice were injected on days 7 and 14 with the E7 vaccine and/or MK-2206 (30 μg). The tumors were measured and the mice euthanized on day 21. A, the combination of MK-2206 with the vaccine had a synergistic effect in the reduction of tumor volume in comparison with the vaccine alone (P < 0.05; combined data from two independent experiments). B, the combination of MK-2206 with the vaccine significantly increased the number of tumor-infiltrating CD8+ T cells in comparison with the nontreated control (P < 0.001; representative example). C, the combination of MK-2206 with the vaccine significantly reduced the number of tumor-infiltrating CD4+Foxp3+ Tregs in comparison with the vaccine alone (P < 0.05; representative example). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 6.
PI3K–Akt inhibition reduces tumor growth in a Treg-dependent manner. Mice were prophylactically treated with wortmannin (WM), triciribine (TCN), or DMSO before s.c. CT26 tumor inoculation. Ex vivo–cultured Tregs were infused in wortmannin-treated mice on day 4 after tumor inoculation. Tumor growth was monitored and the Foxp3+ T-cell infiltrate was then assessed on days 20 and 24. A, prophylactic treatment of the mice with triciribine for 1 week before s.c. tumor inoculation resulted in a significant tumor growth inhibition (P < 0.0001). B, prophylactic treatment of the mice with wortmannin for 1 week before s.c. tumor inoculation resulted in a significant tumor growth inhibition wortmannin (P < 0.0001). This effect was reversed when Tregs were reconstituted by ex vivo–cultured Treg infusion on day 4. C, analysis of FoxP3+ T-cell infiltration into CT26 tumor (days 20 and 24) elucidated a significant decrease in Treg numbers per million tumor cells after wortmannin treatment (P < 0.01), which was restored after the infusion of ex vivo–grown Tregs. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Volume 2, Issue 11
