PVR up-regulation is ATM/ATR dependent and is associated with enhanced H2AX phosphorylation. (A) CFSE-labeled PBMCs were stimulated with SEB. At day 2, cells were treated with caffeine (5mM) or KU-55933 (30μM). After an additional 18 hours, cells were stained with anti–CD3/PECy5, anti-PVR/PE (empty histogram), anti–CD25/allophycocyanin (empty histogram), or control isotypic Ig (filled histogram) and PVR and CD25 expression was analyzed on CD3⁺CFSE^low T cells. One representative experiment of 3 is shown. (B) PBMCs were prepared as described in panel A. The MFI of isotypic control mAb was subtracted from PVR MFI. Data are represented as the mean values of MFI ± SD of PVR on CD3⁺CFSE^low cells of 4 different donors. Statistical analysis was with the Student paired t test. (C) CFSE-labeled PBMCs were stimulated or not with SEB for 3 days and stained with anti–γH2AX mAb plus GAM/PE followed by anti–CD3/allophycocyanin mAb. γH2AX expression on SEB-stimulated T cells was analyzed on CFSE^low and CFSE^high populations. The mean values of MFI ± SD of 7 donors tested are shown. Statistical analysis was with the Student paired t test. (D) CFSE-labeled PBMCs were stimulated with SEB for 3 days and stained with anti-PVR mAb plus GAM/PE followed by anti–γH2AX/Alexa Fluor 647 mAb. The percentage of cells in each quadrant is reported. The 2 donors shown are representative of the 6 analyzed.