Effects of OPN on T-cell activation. C3H.SW CD8⁺ T cells (CD45.2⁺CD8⁺) were injected into lethally irradiated B6/SJL recipient mice accompanied by C3H.SW TCD BM. These recipients were treated with anti-OPN Ab or control IgG at days 0, 3, and 6 after transplantation. Donor-derived CD8⁺ T cells were recovered from spleen of the recipients at day 7 after transplantation, and their levels of CD69, CD25, CD44, and CD62L molecules were measured by flow cytometry (A). In ex vivo experiments, C3H.SW CD8⁺ T cells were stimulated with the indicated reagents. Dose as follows: plate-coated anti-CD3 Ab (10 μg/mL) or soluble anti-CD3 Ab (1 μg/mL), anti-CD28 Ab (2 μg/mL), OPN (10 μg/mL), LY294002 (LY, 20μM). Twenty-four hours after stimulation, expression of the CD69 molecule was measured by flow cytometry (B). Gray shadow and solid line indicate the immunofluorescence intensity of cells as a control and the test of Ab, respectively, and the levels of IL-2 and Bcl-x_L were measured by quantitative real-time PCR (E). Results were normalized to the expression in naive CD8⁺ T cells and expressed as mean plus or minus SEM of 3 cultures. Seventy-two hours after the aforementioned stimulation, T-cell proliferation under the indicated treatments was measured by incorporation of [³H]thymidine (C). Data are mean plus or minus SEM of 4 cultures. Levels of cyclin A, p-Rb, and p27^kip1 in activated CD8⁺ T cells 24 hours after stimulation were determined by Western blot (D). Levels of p-Akt in CD8⁺ T cells at the indicated time points after stimulation were tested by Western blot (F). Quantification was done by densitometry of Western blot bands. Densities were normalized to β-actin, and relative folds were normalized to expression levels in CD8⁺ T cells with control treatment. These results are representative of 3 independent experiments. *P < .05.