Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin

mTOR activity is required for CD3/CD28-induced, IL-2-independent T cell proliferation. Wild-type (A and B) and IL-2^−/− (C and D) DO11.10 TCR transgenic T cells, and control (E and F) and anergic (G and H) A.E7 T cells were labeled with CFSE and left unstimulated (solid line histogram) on immobilized anti-CD3 and anti-CD28 mAbs (dotted line histogram) in the absence (A, C, E, and G) or presence (B, D, F, and H) of RAPA. Representative FACS histograms of CFSE content of viable cells are depicted.

Signaling via mTOR is dispensable for T cell proliferation in the presence of optimal IL-2 amounts. CFSE-labeled A.E7 cells were cultured in plain medium (solid line histogram) or with IL-2 (dotted line histogram) at 0.4, 2, and 10 IU/ml in the absence or presence of RAPA. B, IL-2 was used at 10 IU/ml. Cells were harvested after 5 days (A) or after 3, 4, 5, and 10 days (B) as depicted. Thereafter, the CFSE cellular content was analyzed by flow cytometry. Representative FACS histograms of viable cells from replicate cultures are shown. The experiment was repeated four times with comparable results.

PI3K and mTOR signals differentially regulate CD3/CD28- and IL-2-driven T cell proliferation. CFSE-labeled A.E7 cells were cultured in plain medium (thin line histogram) or stimulated (thick line histogram) with immobilized anti-CD3 and anti-CD28 mAbs (CD3/CD28) (A), IL-2 (0.4 IU/ml) (B), IL-2 (10 IU/ml) (C), or with anti-CD3 and anti-CD28 mAbs and IL-2 (10 IU/ml) (D) in the absence (−) or the presence of LY294002 (LY), RAPA, and a combination of the drugs (LY+RAPA). After 5 days cells were analyzed by flow cytometry. TO-PRO-3 was added at the time of flow cytometry analysis. A–D, Histograms depict the CFSE content of TO-PRO-3⁻ viable cells. The percentage of CFSE^dim cells is indicated in each plot. In the absence of stimulation, 2.3% (inset top left) of the cells appeared as CFSE^dim cells. E, Dot plots of total events are shown. The frequency of cells within each quadrant is indicated. The experiment is representative of four independent experiments.

CD3, CD28, and IL-2 control proliferation of primary T cells via PI3K and mTOR. CFSE-labeled primary DO11.10 lymph node cells were cultured in plain medium (thin line histogram) or stimulated (thick line histogram) with immobilized anti-CD3 mAb (A), with anti-CD3 and IL-2 (10 IU/ml) (B), with anti-CD3 and anti-CD28 mAbs (C), or with OVA_323–339-pulsed irradiated splenocytes (Ag/APC) (D) in the absence (−) or presence of LY294002 (LY), RAPA, or a combination of the drugs (LY+RAPA). After 5 days cells were analyzed by flow cytometry. TO-PRO-3 was added at the time of flow cytometry analysis. A–D, Histograms depict the CFSE content of TO-PRO-3⁻ viable cells. The percentage of CFSE^dim cells is indicated in each plot. In the absence of stimulation, 6.7% (inset top left) of cells appeared as CFSE^dim cells. E, The total number of trypan blue-negative viable cells is shown. F, Dot plots of total events are shown. The frequency of cells is shown in each quadrant. The experiment is representative of four independent experiments.

CD3, CD28, and IL-2 independently control proliferation of primary T cells. DO11.10 primary cells were left untreated (thin line histogram) or stimulated on immobilized anti-CD3 and anti-CD28 mAbs (thick line histogram) for 5 days (A and B) or for 24 h (C and D) and then cultured in their conditioned medium (CM) (A and C) or in fresh medium (FM) (B and D) for an additional 4 days. B and D, The conditioned medium was removed after 24 h and replenished with fresh medium. Representative FACS histograms of CD4⁺ viable cells are depicted. E, The total number of trypan blue-negative viable cells is shown. One representative experiment of two independent determinations is reported.

mTOR-dependent signaling is required for CD3/CD28-induced cyclin D3 and cyclin E expression. A.E7 cells were either left untreated (−) or stimulated with immobilized anti-CD3 and anti-CD28 mAbs (CD3/CD28) or IL-2 (10 IU/ml) in the absence or presence of RAPA or LY294002 (LY). A–C, After 1, 3, and 5 days, cells were recovered and protein extracts were analyzed by SDS-PAGE with anti-cyclin D3, anti-p27^Kip, anti-cyclin E, and anti-actin Abs as indicated. The experiment reported is representative of at least three independent determinations.

mTOR signaling is needed for CD3/CD28-induced cyclin D and cyclin E expression in anergic T cells. Control and anergic A.E7 cells were either left untreated (−) or stimulated with immobilized anti-CD3 and anti-CD28 mAbs (3/28) or IL-2 (10 IU/ml) in the absence or in the presence of RAPA. After 4 days, the cells were recovered and protein extracts were analyzed by SDS-PAGE with anti-cyclin D3, anti-cyclin E, and anti-actin Abs. The experiment shown was repeated once with similar results.