MHC class II tetramers engineered for enhanced binding to CD4 improve detection of antigen-specific T cells.

ABSTRACT

The ability to identify T cells that recognize specific peptide antigens bound to major histocompatibility complex (MHC) molecules has enabled enumeration and molecular characterization of the lymphocytes responsible for cell-mediated immunity. Fluorophore-labeled peptideMHC class I (pMHCI) tetramers are well-established reagents for identifying antigen-specific CD8+ T cells by flow cytometry, but efforts to extend the approach to CD4+ T cells have been less successful, perhaps owing to lower binding strength between CD4 and MHC class II (MHCII) molecules. Here we show that pMHCII tetramers engineered by directed evolution for enhanced CD4 binding outperform conventional tetramers for the detection of cognate T cells. Using the engineered tetramers, we identified about twice as many antigen-specific CD4+ T cells in mice immunized against multiple peptides than when using traditional tetramers. CD4 affinity-enhanced pMHCII tetramers, therefore, allow direct sampling of antigen-specific CD4+ T cells that cannot be accessed with conventional pMHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire.