Cd161 apc clone dx12

PBMC (10⁶ cells/donor) and CMC were stained for ex vivo cellular phenotype using CD3-PE.Cy5 (clone UCHT1; BD Biosciences), CD4-FITC (clone RPA-T4; BD Biosciences), and CD161-APC (clone DX12; BD Biosciences). The cells were also stained for the HIV co-receptor CCR5-V450 (clone 2D7/CCR5; BD Biosciences), and cellular activation markers CD69-PE.Cy7 (clone FN50; BD Biosciences), CD95-PE (clone DX2; BD Biosciences), and HLA-DR-APC.H7 (clone L243; BD Biosciences). Dead cells were eliminated from the analysis using Far Red-Live/Dead discrimination (Invitrogen).
Data were acquired on an LSRII flow cytometer (BD System) and analyzed using FlowJo v10.0.8r1 (TreeStar). Samples were gated on forward scatter height (FSC-H) and forward scatter area (FSC-A) for excluding doublets, then on lymphocytes population with a side scatter area (SSC-A) and FSC-A gating. From the lymphocytes gate, live cells were gated with the far-Red-live dead/SSC-A. CD3⁺ cells were gated from the live cells and CD4⁺ T cells from the CD3⁺ gates (Supplementary Fig. S1). Cervical samples with less than 100 CD3⁺ cells were excluded from the analyses.

For cytometric assessment of MAIT cell phenotype, bulk PBMCs were stained with LIVE/DEAD^TM Fixable Blue Dead Cell Stain Kit (Invitrogen) and Fc receptor blocking reagent (Miltenyi Biotec) and a combination of the following antibodies (from BioLegend except as noted): CD3 BV655 (clone OKT3), CD8 BV711 (clone RPA-T8), CD161 APC (clone DX12, BD Biosciences), Vα7.2 (clone 3C10), CD69 PE (clone FN50), CD107a PerCP-Cy5.5 (clone H4A3), granzyme B Pacific Blue (clone GB11), perforin FITC (clone), and interferon γ APC-Cy7 (clone 4S.B3).

For cytometric assessment of MAIT cell phenotype, bulk PBMCs were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen) or Annexin V Apoptosis Detection Kit with 7-AAD FITC (Biolegend), with Fc receptor blocking reagent (Miltenyi Biotec) and a combination of the following antibodies (from BioLegend except as noted): CD3 BV605 (clone OKT3), CD161 APC (clone DX12, BD Biosciences), Vα7.2 PE-Cy7 (clone 3C10), CD69 PE (clone FN50), CD107a PerCP-Cy5.5 (clone H4A3), GzmB Pacific Blue (clone GB11), perforin FITC (clone), and IFNγ APC-Cy7 (clone 4S.B3). For MR1 expression analysis on antigen presenting cells, bulk PBMCs were stained with Fc receptor blocking reagent (Miltenyi Biotec), LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen), and a combination of the following antibodies (from BioLegend): CD11c BV421 (clone 3.9), CD123 BV510 (clone 6H6), CD14 BV421 (clone 3D3), CD3 BV605 (clone OKT3), CD45 FITC (clone 2D1), CD56 BV711 (clone 5.1H11), CD16 APC (clone 3G8), HLA-DR PE-Cy7 (clone LN3), and MR1 PE (clone 26.5).