Plzf apc

Antibodies used were as follows: CD56 BV421, CD3 PE-Cy7 or APC-Cy7, CD14 APC-Cy7, CD19 APC-Cy7, CD57 Pacific Blue or FITC, CD62L PE-Cy7, CD107a PE-Cy7, CD244 PE-Cy5.5, Perforin Pacific blue, CD160 Alexa Fluor 647 (Biolegend), CD16 eFluor450 or FITC, CD69 FITC, CD94 FITC, CD8a PerCP-Cy5.5 (eBioscience), CD161 PE or APC, CD56 APC, IFNγ FITC, NKp30 APC, NKp46 APC, NKp80 APC (Miltenyi Biotec), CD3 Pacific Orange, CD4 Qdot 605, Granzyme B APC (Invitrogen), NKG2C Alexa Fluor 488 or PE, NKG2D PE, PLZF APC, Granzyme A FITC (R&D Systems), CD56 FITC or PE-Cy7, CD85j FITC, IFNγ Alexa Fluor 700, Ki67 FITC (BD Biosciences), Granzyme K FITC (Immunotools), NKG2A PE, NKp44 PE, CD158e1/e2 PE (Beckman Coulter), Phosphatidylserine Alexa Fluor 488 (Merck Milipore). The viability dye Live/Dead fixable Near-IR (Invitrogen) was used in all experiments. Anti-KLRG1 FITC was kindly provided by H. Pircher. Data were acquired on LSRII (BD Biosciences) and analyzed using FlowJo (Treestar, Inc.).

Cryopreserved PBMC specimens were thawed and washed, and counts and viability were assessed using the Countless Automated Cell Counter system (Invitrogen, Carlsbad, CA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining for IFNγ and promyelocytic leukaemia zinc finger (PLZF) were performed in Perm/Wash (BD Biosciences) at room temperature for 15 min in the dark. mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD69 AF00 (clone FN50de), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCR Vα7.2 PE, TCR Vα7.2 PerCP-Cy5.5 (clone 3C10) were from Biolegend (San Diego, CA, USA), and PLZF APC was from R&D Systems (Minneapolis, MN). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA) and added to the cells together with the cell surface antibodies. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).

Cryopreserved specimens were thawed and washed, and counts and viability were assessed using the Countess Automated Cell Counter system (Invitrogen, Carlsbad, CA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining was performed in Perm/Wash (BD Biosciences). mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD127 FITC (clone HIL-7R-M2), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, PLZF APC was from R&D Systems (Minneapolis, MN), EOMES FITC (clone WD1928) was from eBioscience and TCR Vα7.2 PE (clone 3C10) was from Biolegend (San Diego, CA, USA). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA). Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).