Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding

Tetramers of major histocompatibility complex (MHC) and peptide represent a large technological advance in immunology by allowing direct analysis of antigen-specific T cells without in vitro manipulation. However, these tetramers show a concentration-dependent background binding to nonspecific T cells by interacting with the co-receptor CD8. We therefore produced tetramers of human leukocyte antigen (HLA)-A0201 and peptides, with a mutation (of alanine to valine at position 245, called V245 here) known to alter the interaction with CD8. These mutated tetramers showed a greatly diminished nonspecific binding but retained specific binding, thus allowing accurate discrimination and very efficient immunomagnetic sorting of rare, specific T cells (less than 1%), without requiring the use of monoclonal antibody against CD8. Therefore, alterations of CD8 binding by mutation of the MHC greatly improved the specificity of MHC-peptide multimers, thus providing efficient tools to sort specific human T cells for immunotherapy.