The fluorochrome labeled monoclonal antibodies (mAbs) anti-CD2-FITC, anti-CD3-Alexa 700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-APC Fluor 780, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20 PECy7, anti-CD19-V450, anti-CD20-APCCy7, anti-CD25-PEcy7, anti- CD28-PE, anti-CD28-FITC, anti-CD38-PE, anti-CD39-FITC, anti-CD45-PerCP, anti-CD56-APC, anti-CD57-FITC, anti-CD197-PECy7, anti-HLA-DR, anti-IgM, anti-IgD, anti-Ki67-FITC, anti-Bcl-2-PE, anti-TNF-α-PE, anti-TNF-α-APC, IFN-γ-PcpCy5.5, and IL-2-PECy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-CD45RA-QDOT655 and anti-CD69-FITC mAb were obtained from Invitrogen (Carlsbad, CA). Anti-CD8-Alexa780, anti-CD27-Alexa Fluor 700, and anti-CD38-eFluor 650NC were obtained from E-bioscience (San Diego, CA). Anti-CD24-PEcy7 and anti-CD279-PE (PD-1) were purchased from Biolegend (San Diego, CA).
Human EBV protein and CMV peptide pool of pp65 sequence consisting of 138 peptides (15 mers with 11 amino acid overlaps) was purchased from JPT Peptide Technologies (Berlin, Germany).
All patients were DSA-free with a calculated panel reactive antibody (PRA) ≤20% at enrollment. Patient samples were assessed for donor-specific alloantibody post-transplantation as described previously (18 (link)).