PVR and Nectin-2 are induced on activated T cells in response to SEB stimulation. (A) PBMCs were stimulated with SEB (100 ng/mL) at different times, T lymphocytes were purified by immunomagnetic positive selection, and total RNA was isolated. RT-PCR was performed as described in “RT-PCR.” One representative donor of the 4 analyzed is shown. (B) PVR and Nectin-2 expression was evaluated by performing immunofluorescence and cytofluorimetric analysis by gating CD3⁺ T cells on SEB-stimulated PBMCs at days 2, 3, 5, and 7. MFI of isotypic control mAb was subtracted from PVR or Nectin-2 MFI. Time-course analysis was performed on 4 different donors and the average of PVR MFI ± SD was: day 0: 0 ± 0; day 2: 2 ± 0; day 3: 14 ± 4; day 5: 11 ± 4; and day 7: 5 ± 1. A representative donor is shown. (C) PBMCs were stimulated with SEB and T cells were purified as described in panel A. Western-blot analysis was performed on total cell lysate using an anti–Nectin-2 mAb. Protein loading was normalized using β-actin. Lysates from the K562 and BAF3 cell lines were used as positive and negative controls, respectively (data not shown). (D) PBMCs were stimulated with SEB for 3 days and then stained with an isotypic control mAb (filled histogram) or Nectin-2 mAb (open histogram). Extracellular (EC) and intracellular (IC) staining was performed as described in “Methods.” Nectin-2 expression was analyzed by gating the CD3⁺ T-cell population.