Cxcr3 bv605

Thawed PBMCs were reacted with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove the dead cells. For phenotypic analysis, cells were stained with phycoerythrin (PE)-conjugated Tax_301-309/HLA-A*24:02 tetramer reagents (MBL, Nagoya, Japan) and several fluorescence-conjugate mouse anti-human monoclonal antibodies (mAbs) [CD3-APC-H7, CD8-Pacific Blue, CD45RA-PerCP5.5, CCR7-Alexa647, CD62L-PE-Cy7, CD27-FITC, CXCR3-BV605 (BD Biosciences), and CD95-PE-Cy5 (Biolegend, San Jose, CA)] for 25 min on ice. Stained cells were washed twice and immediately acquired using FACSAriaIII Fusion (BD Biosciences) equipped with 20 detectors by 4-lasers at 488 nm, 561 nm, 633 nm, and 405 nm. The data were analyzed using FlowJo ver.10 software (BD Biosciences). The experiments requiring cell sorting for TCR repertoire analysis, described below, were carried out using the same equipment.

The following fluorochrome-conjugated anti-human mAb were used for flow cytometry studies: LAG3 BV650, TIGIT PE-Cy7, CTLA4 PE, CD25 PE-Dazzle 594, TIM3 PerCP-Cy5.5, CD19 Alexa Fluor 700, CCR4 BV421, ICOS PE-Cy7, CCR5 PE-Dazzle 594, HLA-DR PE, CCR7 FITC, CD38 BV711, PD-L1 BV711, CCR6 Alexa Fluor 700, PD-1 BV421, CD40L BV605, perforin PE-Dazzle 594, and CD8 PerCP from BioLegend (San Diego, CA); CD3 BUV496, CD4 APC-Cy7, CD4 APC-H7, CD127 BV605, Ki-67 PerCP-Cy5.5, PD-1 BV650, CXCR3 BV605, CD69 BV650, IL-2 BV711, CXCR5 Alexa Fluor 647, IFN-γ PE-Cy7, TNF-α FITC, granzyme B Alexa Fluor 700 and CD27 BV480 from BD Biosciences (San Jose, CA); IL-21 PE from eBioscience, (San Diego, CA); and CD45RO PE-Cy5.5 from Beckman Coulter (Fullerton, CA). LIVE/DEAD Fixable Blue Dead Cell Stain Kit from Thermo Fisher Scientific (Boston, MA) was used to detect and exclude dead cells. All the reagents were tested and titrated for optimum concentration before usage.