TCRαβ⁺ DN T cells in healthy individuals. Lymphocytes isolated either from PBMCs or LNs of healthy donors were stained with anti–CD3-allophycocyanin (APC), anti–CD4-peridinin chlorophyll protein (PerCP), anti–CD8-PerCP, and anti–TCRαβ-PE or anti–TCRγδ-FITC mAbs and analyzed by flow cytometry. The cells were gated on DN T lymphocytes via their forward and side scatter properties and their CD3⁺, CD4^-, and CD8^- expression profile. (A) Results represent mean percentages (± SD) of TCRαβ⁺ and TCRγδ⁺ DN T cells within total CD3⁺ T cells isolated either from peripheral blood (▪) or LNs (□). (B-C) CD3⁺ CD4^-CD8^- DN T cells from peripheral blood lymphocytes can be separated in CD3^high TCRγδ and CD3^intermediate TCRαβ T cells. (B) The dot plots show staining with APC-conjugated anti-CD3 mAb and PerCP-conjugated anti-CD4/CD8 mAb. The region R1 indicates the population of CD3^high DN T cells; the region R2 shows CD3^intermediate T cells. (C) Dot plots show staining for anti-TCRαβ or anti-TCRγδ mAbs for cells of R1 (left plot) and R2 (right plot). Numbers in the upper left and lower right quadrants represent percentages of TCRαβ⁺ and TCRγδ⁺ cells within CD3⁺ DN T cells, respectively. One set of representative results of 20 healthy donors is shown.