SEB-activated CD4⁺ and CD8⁺ T cells express NKG2D ligands on their cell surface. (A) PBMCs were CFSE-labeled and then stimulated with SEB. After 3, 5, and 7 days cells were harvested and stained with anti-CD8, anti-CD3, and antibodies specific for NKG2D ligands (MICA, ULBP1, ULBP2, and ULBP3), in a 4-color FACS analysis. Subpopulations of CD8⁺ and CD4⁺ T lymphocytes were identified by gating on CD8⁺ CD3⁺ or CD8⁻ CD3⁺ cells, respectively, and further analyzed for the expression of NKG2D ligands. Progressive loss of CFSE fluorescence intensity in CD4⁺ or CD8⁺ T lymphocytes is indicated by the R1 region. The median of MFI relative to each ligand and calculated on cells gated in R1 (which includes both quadrants to the left) is shown. The percentage of cells in all the quadrants is also indicated. X-axis, CFSE fluorescence intensity. Y-axis, NKG2D ligand fluorescence intensity. A representative donor of 13 tested is shown. (B) MICA, MICB, ULBP1, ULBP2, and ULBP3 transcripts in SEB activated CD3⁺ T cells, quantitated by real-time PCR. PBMCs were stimulated with SEB, cells were harvested at different times, and CD3⁺ T lymphocytes were further purified by immunomagnetic positive selection and total RNA was isolated. Real-time PCR was performed as described in Materials and methods. Data were normalized by the amount of β-actin mRNA. The range of C_T values for each ligand was as follows: 27 to 29 for MICA, 25 to 27 for MICB, 30 to 34 for ULBP1, 30 to 33 for ULBP2, 33 to 35 for ULBP3, and 15 to 18 for β-actin. (C) Reverse-transcriptase PCR analysis of ULBP4 and GAPDH in SEB-activated T cells. The number of PCR cycles was 36 (ULBP4) or 28 (GAPDH).