TLR9 engagement plus γ-radiation enhances cell-cycle checkpoint activation. (A) CD4 T cells were labeled with CFSE (1 μM), activated with plate-bound anti-CD3 and CD28 antibodies for 48 hours. Cells were removed and placed in fresh media containing TLR9 or control ligand (10 μg/mL) for 24 hours followed by IR (300 cGy γ-radiation). Cells were maintained in the presence of IL-2 (50 U/mL) for an additional 3 days. Seventy-two hours after IR, T-cell division was evaluated by flow cytometry. Each CFSE peak, after the first peak, represents one cycle of cell division. (B) CD4 T cells were activated with plate-bound anti-CD3 and CD28 antibodies for 48 hours. Cells were removed and placed in fresh media containing TLR9 or control ligand (10 μg/mL) for 24 hours followed by IR (300 cGy γ-radiation). Twenty-four hours after IR, cells were harvested, stained with propidium iodide, and analyzed by flow cytometry. The data in the histogram in panel B show the DNA content of cells from different groups. The percentage of cells in the G-2M phase, compiled from several experiments (± SD) is shown in panel C. P values were determined by use of Student t test; P < .005. (D) CD4 T lymphocytes were treated as described in panel C except that protein was collected from TLR-stimulated or nonstimulated cells plus or minus IR 20 minutes after IR. Western blot was used to analyze the indicated proteins. (E) CD4 T lymphocytes were treated as described in Figure 1. Before IR exposure, UCN-01 or DBH, which inhibit Chk1 and Chk2 activation, or wortmannin, which inactivates PI3K activity, were added to cell cultures. The levels of apoptotic cells as determined by annexin V and propidium iodide staining were evaluated 24 hours after IR and were measured by flow cytometry. The data shown is representative of at least 2 experiments (± SD), each showing identical results. The Student t test was used to determine P values between the indicated groups and irradiated, TLR9-treated cells; *P < .05.