Cells were labeled with the following antibodies according to the manufacturer's recommendations: allophycocyanin (APC)-conjugated CD19 (CD19-APC) (clone PeCa1; ImmunoTools), CD4-APC/Cy7 (clone GK1.5), CD44-APC/Cy7 (clone IM7), CD8a-Pacific Blue (clone 53.6.7), NK1.1-APC (clone PK136), PD-1 conjugated with fluorescein isothiocyanate (FITC) or brilliant violet 421 (clone 29F.1A12) (all from BioLegend), and CD8a-FITC (clone 1D3; eBioscience).
For pentamer staining. 2 × 106 isolated cells were incubated for 10 min at room temperature (RT) with major histocompatibility complex (MHC) class I peptide pentamers (H-2Kb/ILSPFLPLL for HBsAg [epitope S208–216], H-2Kb/MGLKFRQL for HBcAg [epitope C93–100], or H-2Kb/SIINFEKL for ovalbumin [OVA] [epitope OVA257–264]; ProImmune). Cells were then washed and were stained with the respective surface antibodies according to the manufacturer's recommendations.
Data were acquired with an 8-color BD FACSCanto II system equipped with three lasers (488 nm, 633 nm, and 405 nm) or with a 4-color FACSCalibur system equipped with two lasers (argon [488 nm] and diode [635 nm]). Fluorescence-activated cell sorter (FACS) data were analyzed with FlowJo or DIVA software.
For pentamer staining. 2 × 106 isolated cells were incubated for 10 min at room temperature (RT) with major histocompatibility complex (MHC) class I peptide pentamers (H-2Kb/ILSPFLPLL for HBsAg [epitope S208–216], H-2Kb/MGLKFRQL for HBcAg [epitope C93–100], or H-2Kb/SIINFEKL for ovalbumin [OVA] [epitope OVA257–264]; ProImmune). Cells were then washed and were stained with the respective surface antibodies according to the manufacturer's recommendations.
Data were acquired with an 8-color BD FACSCanto II system equipped with three lasers (488 nm, 633 nm, and 405 nm) or with a 4-color FACSCalibur system equipped with two lasers (argon [488 nm] and diode [635 nm]). Fluorescence-activated cell sorter (FACS) data were analyzed with FlowJo or DIVA software.