Bv786 labeled pd 1

Fresh tumor digests (FTD) from tumor tissues were prepared using the gentleMACS tumor dissociator (Miltenyi Biotec, Auburn, California, USA), according to the manufacturer’s instructions, and were cryopreserved until use. Cryopreserved FTD were thawed in Roswell Park Memorial Institute (RPMI) 1640 mediumand then stained after blocking Fc receptors using Human BD Fc Block (BD BioSciences, San Jose, California, USA). The following monoclonal antibodies were used: Brilliant Violet (BV)421-labeled CD3, Allophycocyanin (APC)-labeled CD4, Fluorescein isothiocyanate (FITC)-labeled CD8, BV711-labeled CD103, Phycoerythrin(PE)-labeled CD39, BV786-labeled PD-1, BV650-labeled Tim-3 and PE-labeled CD31 (all from BioLegend, San Diego, California, USA), and APC-labeled fibroblast activation protein (FAP) (R&D Systems, Minneapolis, Minnesota, USA). SYTOX AADvanced Dead Cell Stain Kit (Thermo Fisher, Waltham, Massachusetts, USA) or Zombie NIR Fixable Viability Kit (BioLegend) was used to exclude dead cells. Stained cells were analyzed using an LSRFortessa flow cytometer (BD BioSciences) and the data processed using FlowJo V.10.0.7 (FlowJo, LLC, Ashland, Oregon, USA). Gating strategies in this study are shown in online supplemental figure S2.

Fresh tumor digests (FTD) were prepared using the gentleMACS tumor dissociator (Miltenyi Biotec, Auburn, California, USA), according to the manufacturer’s instructions. Cell counting was performed by manually using a hemocytometer. FTD (2–5×10⁶ cells/tube) were cryopreserved with CP-1 (Kyokutoseiyaku, Tokyo, Japan) and kept in a deep freezer (−135°C) until use. Cryopreserved FTD (2–5×106 cells) were thawed in RPMI and then stained after blocking Fc receptors using Human BD Fc Block (BD Biosciences, San Jose, California, USA). The following monoclonal antibodies (mAbs) were used: BV421-labeled CD3, APC-labeled CD4, FITC-labeled CD8, BV711-labeled CD103, PE-labeled CD39, and BV786-labeled PD-1 (all from BioLegend, San Diego, California, USA). SYTOX AADvanced Dead Cell Stain Kit (Thermo Fisher, Waltham, Massachusetts, USA) or Zombie NIR Fixable Viability Kit (BioLegend) was used to exclude dead cells. Stained cells were analyzed using an LSRFortessa X-20 flow cytometer (BD Biosciences) and data processed using FlowJo V.10.0.7 (BD Biosciences).