A rapid method for the isolation of untouched naive CD8 T cells from peripheral blood mononuclear cells (46.9)

Abstract Naïve CD8 T cells are, by definition, those that have been released by the thymus into circulation, but have not yet encountered their cognate antigens. Once they become activated they proliferate and generate effector T cells. We describe an easy and rapid method for the isolation of untouched naïve CD8 T cells from peripheral blood mononuclear cells (PBMC). Briefly, PBMCs are isolated by Ficoll-Paque PLUS density gradient sedimentation. Naïve CD8 T cells are then enriched using immunomagnetic, column-free, negative selection (EasySep™). Using EasySep™, unwanted cells are removed using antibody complexes recognizing non-naïve CD8 T cells and dextran-coated magnetic particles. Labeled cells are separated by placing the tube in an EasySep™ magnet. This procedure can be fully automated using RoboSep™. EasySep™ isolated naïve CD8 T cells are assessed by flow cytometry using a combination of antibodies specific for naïve, effector and memory cells. Naïve CD8 T cells are defined as CD8+CD45RA+CCR7+ and CD45RO-CD56-CD57-. Starting with PBMCs isolated from fresh blood and buffy coat, purities of 89±3% (n=8) are obtained (starting cell frequency 1.0-11.2%). EasySep™ isolated naïve CD8 T cells undergo robust proliferation as measured by flow cytometry of CFSE-labeled cells in culture with CD3/28 T cell expander beads. This simple method to isolate naïve CD8 T cells will be invaluable to researchers studying T cell differentiation, autoimmunity and infectious diseases.