Cd8 apc cy7 sk1

Cryopreserved PBMCs were thawed and re-suspended in cold FACS buffer (PBS, 2% bovine serum, 2mM EDTA). CD8⁺ T-cells were enriched by negative selection (Miltenyi Human CD8 negative selection kit, Ref. 130-096-495), and were incubated for 15 min with either HLA-B07:02/RPIDDRFGL-dextramers (day 15 and day 136 cells) or HLA-A02:01/LLWNGPMAV-dextramers (day 15 cells) (Immudex, Denmark), followed by incubation with a panel of surface antibodies to detect live CD3⁺CD8⁺ T-cells (CD3-Alexa Flour 700 (UCHT1, BD Biosciences), CD8-APC Cy7 (SK1, BD Biosciences), CD4-PE-Cy5 (RPA-T4, Ebiosciences), CD14-Horizon V500 (MΦP9, BD Biosciences), CD19-Horizon V500 (HIB19, BD Biosciences), and Live/Dead Fixable Aqua dead cell stain kit (Invitrogen, Cat. L34957)). After 30 minutes of incubation at 4°C, the cells were washed and resuspended in cold FACS buffer for sorting. The cells were sorted using single-cell mode on a BecktonDickinson ARIA III cell sorter (equipped with a 405nm violet laser, 488nm blue laser, 561nm yellow/green laser, and 633nm red laser) into PCR plates containing 4ul Smart-seq2 lysis buffer.

Cryopreserved PBMCs from IRB consented blood donors were thawed at 37°C, washed twice with complete RPMI media [cRPMI; RPMI 1640 medium (Cellgro) supplemented with 5mM HEPES, 2mM Glutamax (Invitrogen), 50 μg/mL Penicillin (Invitrogen), 50 μg/mL Streptomycin (Invitrogen), 50 M 2-mercaptoethanol (Sigma-Aldrich), and 10% FBS (Atlanta Biologicals)], and plated at 2.5×10⁵ cells per well in cRPMI. Cells were activated with soluble anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL), and treated with IL-12 (1 ng/mL) and/or IL-18 (10 ng/mL) (R&D Systems) for 72h as described previously^{1 (link)}. GolgiStop (4 μL/6 mL culture; BD Biosciences) was added four hours prior to staining for flow cytometric analysis. Cells were first stained for the following surface markers: CD4–Pacific Blue (RPA-T4; eBioscience), TIGIT–APC or –PerCP-eFluor710 (MBSA43; eBioscience), CD226–PE (11A8; BD Biosciences), CD8–APC-Cy7 (SK1; BD Biosciences), and CD25–APC or –AlexaFluor (AF)-488 (BC96; BioLegend). For intracellular staining, cells were fixed, permeabilized, and stained for IFN-γ–PE-Cy7 (4S.B3; BioLegend), Helios–PE, –Pacific Blue, or –AF647 (22F6; BioLegend), and FOXP3–AF488 or –PE (206D; BioLegend) using the FOXP3 Fix/Perm staining kit (BioLegend) according to manufacturer’s protocol. Samples were collected on a BD LSRFortessa and analyzed using FlowJo software.