Anti cd45 apc h7 2d1

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.

The following antibodies and reagents were used for the analysis of synovial cells with flow cytometry and cell sorting: anti-CD45-APC-H7 (2D1, BD Pharmingen), anti-CD235a-APC-Alexa Fluor750 (11E4B-7-6(KC16), Beckman Coulter), anti-CD31-PE-Cyanine7 (WM-59, eBioscience), anti-CD146-BV450 (P1H12, BD Horizon), anti-CD34-PE (4H11, eBioscience), anti-PDPN-PerCP-eFluor710 (NZ-1.3, eBioscience), anti-THY1-FITC (5E10, BD Pharmingen), anti-cadherin-11-biotin (23C6), human TruStain FcX (BioLegend), streptavidin-APC (Jackson ImmunoResearch), Live/Dead fixable aqua dead cell stain kits (Molecular Probes). For immunofluorescence staining of synovial tissue, following antibodies and reagents were used: anti-CD45 (135-4C5, AbD Serotec), anti-CD34 (EP373Y, Abcam), anti-PDPN (NZ-1.3, eBioscience), anti-THY1 (F15-42-1, Merck Millipore, and clone Thy-1A1, R&D Systems), anti-cadherin-11-Biotin (23C6), anti-Ki67 (16A8, BioLegend), anti-mouse IgG1-FITC (Southern Biotech), anti-mouse IgG2a FITC (Southern Biotech), anti-mouse IgG2b-Alexa Fluor 647 (Life Technologies), anti-rat IgG-Alexa Fluor 594 (Life Technologies), anti-rat IgG-Alexa Fluor 647, anti-rabbit IgG-Alexa Fluor 546 (Life Technologies), Hoechst 33258 (Life Technologies), and anti-FITC Alexa Fluor 488 (Life Technologies).

The following antibodies and reagents were used for the analysis of synovial cells with flow cytometry and cell sorting: anti-CD45-APC-H7 (2D1, BD Biosciences, CA, USA), anti-CD235a-APC-Alexa Fluor750 (11E4B-7-6, Beckman Coulter, FL, USA), anti-CD31-PE-Cyanine7 (WM-59, eBioscience, CA, USA), anti-CD146-APC (P1H12, eBioscience), anti-CD34-PE (4H11, eBioscience), anti-PDPN-PerCP-eFluor710 (NZ-1.3, eBioscience), anti-THY1-FITC (5E10, BD Bioscience), anti-CD73-PE-CF594 (AD2, BD Bioscience), anti-CD271-APC (ME20.4, eBioscience), anti-CD54-PE-CF594 (HA58 BioLegend, CA, USA), anti-CD44-APC (G44-26 BD Bioscience), anti-CD29-APC (TS2/16 BioLegend), human TruStain FcX (BioLegend), and Live/Dead fixable aqua dead cell stain kits (Molecular Probes, Thermo Fisher Scientific, MA, USA).