Anti cd69 apc clone fn50

10 million KG-1 cells were labelled with CFSE according to manufacturer’s protocol (Thermo Fisher Scientific). The KG-1 cells were then pulsed with 10 μM HIV peptide for 3 hours at 37 °C, 5% CO₂. The KG-1 cells were resuspended at 5×10⁵ cells/mL and aliquoted into 96-well plate at 200 μL per well. The KG-1 cells were washed once to remove excess peptides. The library of 10 million T cells were resuspended at 5×10⁵ cells/mL and aliquoted into the 96-well plate with KG-1 cells at 200 μL per well. After 14-hour activation, the cells were stained with anti-CD69-APC (clone FN50, BioLegend) and B35-HIV-PE tetramer (the method of making pMHC tetramer is described below) on ice for 30 min. Cells were sorted to select tetramer-staining-low (comparable to TCR55 WT T cell’s tetramer staining), anti-CD69-staining-high (top 5% in terms of anti-CD69 MFI) population. Cells were sorted into FBS to maintain cell health. Sorted cells were cultured in cRPMI. It took 2 weeks to grow enough cells to continue the next round of selection. After 3–5 rounds of selection, single cell clones were obtained by diluting cells to 2.5 cells/mL and aliquoting 200 μL cell dilution to each well of 96-well U-bottom plate (Corning). It took 2–4 weeks to grow enough number of cells from single cell clone. Each single cell clone was tested by TCR55 signaling assay described below.