Anti ccr7 apc cy7 clone g043h7

Cryopreserved tonsil cells and PBMCs were thawed quickly, resuspended in complete medium in the presence of DNase I (10 U/ml; Sigma-Aldrich), and rested at 1 × 10⁶ to 2 × 10⁶ cells per well in 96-well U-bottom plates (Corning) for 5 hours at 37°C. The medium was then supplemented with the relevant peptide pools (0.5 μg/ml per peptide). Negative control wells contained equivalent DMSO, and positive control wells contained SEB (0.5 μg/ml; Sigma-Aldrich). After 18 hours, cells were washed in FACS buffer and stained with anti–CCR7–APC-Cy7 (clone G043H7; BioLegend) for 10 min at 37°C. Additional surface stains were performed for 30 min at room temperature in the presence of Brilliant Stain Buffer Plus (BD Biosciences). 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 1% PFA (Biotium), and acquired using a FACSymphony A5 (BD Biosciences). Flow cytometry reagents are listed in table S4.

Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated whole blood by using Hypaque-Ficoll (GE Healthcare) density gradient centrifugation and then cryopreserved in fetal bovine serum (Sigma-Aldrich) containing 10% DMSO (Sigma-Aldrich). The flow cytometry panel was tested on all study subjects within 1-month time interval to avoid intra- and interindividual differences of the analysis. The following antibodies were used: anti-CD14 V500 (clone M5E2), anti-CD19 V500 (clone HIB19), anti-CD152 (CTLA-4) (clone BNI3), anti-Helios AF488 (clone 22F6), anti-HLA-DR BV605 (clone G46-6) (BD Bioscience); anti-CD3 BV570 (clone UCHT1), anti-CD8 BV711 (clone RPA-T8), anti-CD25 PE-Cy5 (clone BC96), anti-CD38 A700 (clone HIT2), anti-CD27 BV785 (clone O323), anti-CCR7 APC-Cy7 (clone G043H7), and anti-PD-1 BV421 (clone EH12.2H7) (BioLegend); anti-CD45RO ECD (clone UCHL1) (Beckman Coulter); anti-CD4 PE-Cy5.5 (clone SK3), anti-FoxP3 PE (clone PCH101), and anti-Tigit PE-Cy7 (clone MBSA43) (eBioscience). LIVE/DEAD Aqua amine dye (Life Technologies) was used to discriminate dead cells and debris.

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.