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

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 peptide:MHC class I (p:MHCI) 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 p:MHCII 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 p:MHCII tetramers, therefore, allow direct sampling of antigen-specific CD4⁺ T cells that cannot be accessed with conventional p:MHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire.