Phenotypic analyses of freshly isolated peripheral TCRαβ⁺ DN T cells. PBMCs from healthy donors were stained with anti–CD3-APC, anti–CD4-PerCP, anti–CD8-PerCP, and anti–TCRαβ mAbs and combinations of mAbs against surface Ag and intracellular proteins. (A) Cells were gated for CD3⁺ CD4^-CD8^- DN T cells. The right 8 dot plots show staining with FITC/PE-conjugated anti-TCRαβ⁺ mAbs and FITC/PE-conjugated mAbs against CD27, CD28, CD25, CD95, CD16, CD56, and intracellular proteins (granzyme B, perforin). (B) CCR7/CD45RA phenotype of TCRαβ⁺ DN T cells. Numbers in the quadrants are the percentages of TCRαβ⁺ DN T cells with the corresponding phenotype. Representative data from one of 10 healthy donors are shown.