Pe hla a2

Cells from PB samples collected by retroorbital bleeding or BM samples were treated with ACK lysis buffer and stained with these dye-conjugated mAbs: Pacific Orange-Ly-6G/6C (Gr-1; Life Technologies), PE-Cyanin5-CD45R/B220, Alexa Fluor 700-CD4, Alexa Fluor 700-CD8a, PE-Cyanin7-CD45.1, APC-cyanin7-CD45.2, Pacific Blue-H2Kd, FITC-H2Kk, FITC-H2Kb, PE-H2Kb, Alexa Fluor 647-H2Kq, Brilliant Violet 510 Streptavidin (BioLegend), Alexa Fluor 700 Sca-1, and APC c-Kit (eBioscience). Data were acquired by FACSAria and analyzed using FlowJo software (Tree Star). Complete blood counts were obtained with MEK-6450 Celltac-α (Nihon Kohden). For flow cytometry analysis of human cell engraftment in NOG mice, BM samples were stained with these dye-conjugated mAbs: PE-HLA-A2, Pacific Blue-human CD45, FITC-conjugated anti–human lineage antibody cocktail, APC-CD34, APC-CD4, APC-CD8, PE-Cyanin7-mouse CD45 (BioLegend), PE-CD33, FITC-CD19 (BD), and PE/Cyanine7-CD38 (eBioscience).

The levels of HLA-A2 expression on PBMCs and HLA-DR, CD83, and CD86 expression on DCs were detected by flow cytometry performed in accordance with the instructions from BioLegend (San Diego, CA, USA). Briefly, 1×10⁶ cells/mL suspended in PBS were incubated with the following anti-human antibodies (2 µg/10⁶ cells): PE-HLA-A2, FITC-CD83, PE-CD86, and APC-HLA-DR (BioLegend). The cells were analyzed at room temperature in the dark for 30 min using the FACS Calibur software (Becton Dickinson, Franklin Lakes, NJ, USA). Fluorescein-conjugated isotype-matched unrelated antibodies were used as negative controls.