γδ T lymphocytes producing IFN-γ and IL-17 express CD161. PBMCs from HIV-1–infected Pts (n = 20) or Hds (n = 10) were stained with the PE-conjugated A13 or BB3 mAbs to identify Vδ1 or Vδ2 T cells, respectively, PerCP-conjugated anti-CD3, and FITC-conjugated anti-CD161 (A-D). Then cells were fixed and permeabilized and cytoplasmically stained with APC-anti–IL-17 and PE-Cy7-anti–IFN-γ was performed on cells before or after 7 days of culture with Ca or PPD and rIL-2 was added on day 4 (B,C: Pts; D: Hds). Samples were analyzed by FACSCanto flow cytometer (Becton Dickinson) gated on CD3/Vδ1 or CD3/Vδ2 cells (A) and on CD161⁺ or CD161⁻ Vδ1 T cells (C,D). (A) Percentage of CD161⁺ cells on CD3/Vδ1 or CD3/Vδ2 gated cells as indicated. (B) One representative case of 10 analyzed: PE-Cy7–IFN-γ/APC–IL-17 staining of CD161⁺ (Bii) or CD161⁻ (Biii) gated cells among Vδ1 T cells (Bi, indicated by []). Results are expressed as log red fluorescence intensity (x-axis, au) versus log green fluorescence intensity (y-axis, au; Bi) or as log far red fluorescence intensity (x-axis, au) versus log very far red fluorescence intensity (y-axis, au; Bii,iii). In panels C-D, results are expressed as percentage of IFN-γ⁺IL-17⁺ cells among CD161⁺Vδ1 or CD161⁻Vδ1 T cells and are the mean ± SD from 20 Pts (C) or 10 Hds (D). *P < .01 versus cells cultured in the absence of antigens (−). **P < .01 versus Hds.