Pe cy7 anti nkg2a z199

Viably frozen PBMCs obtained from 8 macaques prior to immunization, post-2^nd Ad5hr-recombinant immunization, and 2 wpi and 12 wpi were thawed and stained (1 ul 5-OP-RU tetramer-PE/1 million cells) for 30 minutes at RT, magnetically labeled with anti-PE magnetic beads (Miltenyi Biotec), and positively isolated using a magnetic column. This subset was considered enriched MAIT cells (Fig. 4). MR1⁻ cells were labeled with anti-CD3 magnetic beads (Miltenyi Biotec), isolated using a magnetic column and considered CD3⁺MR1⁻ T cells. MR1⁻CD3⁻ cells were labeled with anti-CD19 magnetic beads (Miltenyi Biotec), isolated using a magnetic column and considered B cells. To check the purity of the enriched MAIT cells, PBMC obtained prior to immunization from 5 additional macaques were stained with 5-OP-RU tetramer conjugated with PE (NIH Tetramer Core Facility) for 30 minutes; followed by Alexa700 anti-CD3 (SP34-2), BV711 anti-CD4 (L200), BV650 anti-CD8 (RPA-T8), APC-Cy7 anti-CD20 (2H7), BUV395 anti-CD123 (7G3), BV421 anti-CD11b (ICRF44) BV786 anti-CD45 (D058-1283) all from BD Biosciences, FITC anti-CD66_abce (TET2) from Miltenyi Biotec, PE-Cy7 anti-NKG2A (Z199) from Beckman Coulter. The cells were acquired on a Symphony (BD Biosciences) and analyzed as described above.

The following monoclonal antibodies were used for flow cytometric analysis: APC anti-NKG2C (REA205) (Miltenyi Biotec; Bergisch Gladbach, Germany), Alexa Fluor 700 anti-KIR2DL1/2DS5 (143211) (R&D Systems), PE-Cy7 anti-NKG2A (Z199) (Beckman Coulter; Brea, CA, USA), BV605 anti-KIR2DL2/2DL3/2DS2 (CH-L), V450 anti-IFN-γ (B27) (BD Biosciences), BV650 anti-CD56 (HCD56), BV785 anti-CD3 (SK7), APC/Cyanine7 anti-CD20 (2H7), PE anti-KIR3DL1 (DX-9), BV510 anti-CD107a (H4A3), PE anti-HLA-E (3D12) (Biolegend). Dead cells were excluded using LIVE/DEAD fixable near-IR dead cell stain kit (Thermo Fisher Scientific; Waltham, MA, USA). After staining PBMCs with LIVE/DEAD Fixable Dead Cell Staining kits for 10 min at room temperature, they were washed and stained with mixed monoclonal antibodies for 30 min on ice and then fixed with 1.6% formaldehyde. For intracellular staining of IFN-γ, PBMCs were fixed and permeabilized by incubation with BD Cytofix/Cytoperm (BD Biosciences) for 20 min on ice, washed using BD Perm/Wash (BD Biosciences), stained with anti-IFN-γ antibody for 30 min on ice, and washed and resuspended in PBS containing 2% FBS. PBMCs were analyzed by using a BD FACSAria Fusion Flow Cytometer (BD Biosciences). Flow cytometry data was analyzed using FlowJo software ver. 10 (Tree Star; Ashland, OR, USA).