Cryopreserved Tonsil Cell Phenotyping

Cryopreserved tonsil cells were thawed quickly, washed in PBS, distributed at 1 × 10⁶ to 2 × 10⁶ cells per well in 96-well U-bottom plates (Corning), and stained with anti–CCR7–APC-Cy7 (clone G043H7; BioLegend) for 10 min at 37°C. Cells were then labeled with a mix of BV421-conjugated human leukocyte antigen (HLA) class I tetramers for 15 min at room temperature in the presence of dasatinib (50 nM; STEMCELL Technologies). The following tetramers were used in these experiments, each corresponding to a defined epitope derived from EBV: GLCTLVAML/HLA-A*02:01, TYGPVFMCL/HLA-A*24:02, RPPIFIRRL/HLA-B*07:02, and RAKFKQLL/HLA-B*08:01 (56 (link)). Additional surface stains were performed for 30 min at 4°C. Viable cells were identified by exclusion using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Stained cells were washed in FACS buffer, fixed in PBS containing with 1% PFA (Biotium), and acquired using a FACSymphony A5 (BD Biosciences). Flow cytometry reagents are listed in table S4.