Flow cytometry and antibody and MHC pentamer labeling.

For surface staining, cells were incubated with anti–human FcR-block (Miltenyi Biotec, 130-059-901), live/dead (Zombie NIR/Aqua, BioLegend, 423106 and 423102), and chemokine-receptor antibodies (listed below) for 15 minutes at room temperature. Cells were subsequently incubated with surface marker antibodies for 30 minutes at 4°C. For intracellular and intranuclear labeling, surface staining was followed by fixation and permeabilization with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, 00-5523-00) according to manufacturer instructions. Antibodies for intracellular targets were incubated for 1 hour at room temperature. All samples were acquired using DIVA software (BD Biosciences) on LSRFortessa/FACSymphony (BD Biosciences) instruments, and analysis was performed using FlowJo software (Tree Star Inc.).

Antibodies used in this study include the following: CD3 (UCHT1, BD Biosciences, BUV661), CD4 (SK3, BD Biosciences, BUV469 / S3.5, Thermo Fisher Scientific, PE-Cy5.5), CD8 (SK1, BioLegend, BV650), CD19 (HIB19, BioLegend, PE-Cy5), CD27 (LG.3A10, BioLegend, BV650), CD39 (A1, BioLegend, BV711), CD45 (HI30, BD Biosciences, BUV395), CD45RA (HI100, BioLegend, APC-Fire750/BV785), CD62L (DREG-56, BioLegend, PE-Cy7/SK11, BD Biosciences, BV510), CD69 (FN50, BioLegend, BV421), CD103 (Ber-ACT8, BioLegend, BV711), CD107a (H4A3, BD Biosciences, FITC), CD127 (IL-7R) (A019D5, BioLegend, Alexa Fluor 700/PE-Dazzle 594), CD279 (PD-1) (EH12.1, BD Biosciences, BUV737), CD335 (NKp46) (9E2, BioLegend, BV510), CD56 (B159, BD Biosciences, APC), Blimp-1 (IC36081R, R&D, Alexa Fluor 647/6D3, BD Biosciences, PE-CF594), CCL5 (VL1, BioLegend, PE), granzyme B (GB11, BioLegend, PE-CF594/Alexa Fluor 700), HLA-DR (G46-6, BD Biosciences, PE-CF594/BV605), IFN-γ (4S.B3, BD Biosciences, BV786), KLRG1 (13F12F2, eBioscience, Alexa Fluor 488), Perforin (dG9, BD Biosciences, PerCP-Cy5.5), TNF-α (Mab11, BD Biosciences, PE-Cy7), anti–mouse TCR-β (H57-597, BioLegend, BV510/605)

EBV-pentamers specific for BMLF1 (GLCTLVAML, HLA-A*02:01, PE/APC) and LMP2 (CLGGLLTMV, HLA-A*02:01, APC/PE) were purchased from Proimmune. Pentamers were added 15 minutes prior to surface antibody labeling during FcR-blocking and live/dead staining.