Apc conjugated anti cd8 rea734 mab

A chimeric protein consisting of a mouse Immunoglobulin G (IgG) fused with two A*02:01 molecules commercialized under the name of DimerX (BD Biosciences) was used to detect CD8 T cells recognizing A*02:01‐restricted epitopes as described.³² Briefly, DimerX was passively loaded with individual A*02:01‐restricted HRV peptides at 640 molar excess in PBS pH 7.2 at 37°C overnight. Peptide‐expanded PBMCs from A*02:01‐donors were then stained with peptide‐loaded DimerX for 1 hour at room temperature (RT) and cognate CD8 T cells were detected by flow cytometry (FACScalibur, BD Biosciences) using PE‐conjugated anti‐mouse IgG1 A85‐1 mAb (BD biosciences) and APC‐conjugated anti‐CD8 REA734 mAb (Miltenyi Biotec). The same PBMCs stained with unloaded DimerX or with DimerX loaded with the A*01:01‐restricted peptide HRVA_2029‐2037 (YGDDVIFSY) were used as negative controls.

PBMCs from matching donors were cultured for 14 hours in RPMI 1640 (Gibco, NY, USA) supplemented with 10% human serum (Gibco NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM l‐glutamine (Lonza, Walkersville, USA) at 37°C and 5% CO₂ with the HRV peptides (10 μM) in the presence of Brefeldin A (5 μg/mL) (Thermo Fisher Scientific). The same PBMCs cultured with media alone were used as negative control. CEF peptide pool (Mabtech) was used as a positive control. After culture, cells were washed with PBS and surface stained with APC‐conjugated anti‐CD8 REA734 mAb (Miltenyi Biotec) followed by intracellular staining with FITC‐conjugated anti‐IFN‐γ 45‐15 mAb (Miltenyi Biotec) according to the manufacturer's instructions. Stained cells were detected by flow cytometry (FACScalibur, BD Biosciences).