Anti cd4 pe cy7 rpa t4

Whole-blood fluorescence-activated cell sorting (FACS) for CD3, CD8, HLA-DR and Ki-67 expression was performed at LabCorp central laboratory according to established protocol. PBMCs were isolated at Precision Bioservices, and cryopreserved samples were shipped to Genentech for analysis of fractalkine receptor expression and detection of tumour-specific T cells. In brief, PBMCs were thawed and rested overnight, then a small aliquot of cells were stained with anti-HLA-A2-FITC (BB7.2, BD) anti-CD45-APC-H7 (2D1, BD) to determine HLA-A2 status. The remaining cells were stained with a mixture of HLA-A*0201/peptide dextramers and pentamer (Immudex and Proimmune, Supplementary Table 4) for 10 min at room temperature followed by anti-CD3-BV510 (UCHTI, Biolegend), anti-CD8-A700 (RPA-T8, BD), anti-CD4-PE-Cy7 (RPA-T4, eBioscience), anti-CD45RA-eVolve605 (HI100, eBioscience), anti-CCR7-BV421 (G043H7, Biolegend), anti-CX3CR1-PerCP-eFluor710 (2A9-1, eBioscience) and Fixable Viability Dye eFluor780 (eBioscience) for 30 min on ice. Samples were washed twice before data acquisition and sorting on a BD FACS Aria running FACSDiva v8 software. A minimum of 10 dextramer-positive events out of 50,000 CD8⁺ T cells is considered a tumour-specific response.