TLR9-stimulation on CD4 T lymphocytes represses IR-induced apoptosis. (A) CD4 T cells were purified by negative selection and then followed by positive selection using magnetic beads. 106 cells were activated with plate-bound anti-CD3 and CD28 antibody for 48 hours. After this time, cells were cultured in fresh media in the absence or presence of TLR9 ligand (10 μg/mL CpG-ODN) for 24 hours, as described in “Methods.” After activation, cells were irradiated (300 cGy γ-radiation) and parked in culture for an additional 24 hours. Cells were then harvested and total cell lysates were used for analysis by Western blot for the expression of indicated proteins. (B,C) Alternatively, 24 hours after irradiation apoptosis was evaluated by staining cells with annexin-V plus propidium iodide and the average of several experiments is shown in panel C. The percentages indicated on the graph are the percent of double positive PI- and annexin V–stained cells (± SD, n = 3 experiments). P values were determined by use of Student t test; P < .02. (D) CD4 T cells were treated as described in panel A. Twenty-four hours after irradiation cell lysates were used to analyze the expression of the indicated proteins by Western blots.
