Analysis of circulating immune cell phenotypes was performed both prior to transplant and at regular intervals following transplantation. Cell frequencies from flow cytometric analysis were combined with complete blood counts to calculate total numbers of circulating T cells and various T cell subsets. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (BD Biosciences, Franklin Lakes, NJ) within 6 hours of phlebotomy. PBMCs (1.5×10^6) were then incubated with antibody mixtures at the appropriate titer for 15 minutes and washed twice. For assessment of intracellular markers, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s direction following surface staining. Flow cytometric data was acquired immediately using a BD LSR II multicolor flow cytometer (BD Biosciences). All flow data was analyzed using FlowJo (Tree Star, San Carlos, CA).
Surface markers were stained with the following monoclonal antibodies (mAbs): CD3 PacBlue, CD3 APC-Cy7, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD127 PE-Cy7, PD-1 APC, LFA-1 APC, CD20 APC-Cy7 (all BD Biosciences), CD95 PacBlue, CD69 FITC (Invitrogen, Grand Island, NY), and CD25 PE (Miltenyi Biotech, San Diego, CA). Intracellular staining for FoxP3 was performed using FoxP3 Alexa488 (Biolegend, San Diego, CA).
Surface markers were stained with the following monoclonal antibodies (mAbs): CD3 PacBlue, CD3 APC-Cy7, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD127 PE-Cy7, PD-1 APC, LFA-1 APC, CD20 APC-Cy7 (all BD Biosciences), CD95 PacBlue, CD69 FITC (Invitrogen, Grand Island, NY), and CD25 PE (Miltenyi Biotech, San Diego, CA). Intracellular staining for FoxP3 was performed using FoxP3 Alexa488 (Biolegend, San Diego, CA).