Braendstrup, Peter1; Justesen, Sune Frederik Lamdahl3; Osterbye, Thomas2; Nielsen, Lise Lotte Bruun4; Mallone, Roberto4; Vindeløv, Lars1; Stryhn, Anette4; Buus, Søren5
1 Hæmatologisk Klinik, Finsencentret, Rigshospitalet, The Capital Region of Denmark2 Patologiafdelingen, Diagnostisk Center, Rigshospitalet, The Capital Region of Denmark3 Department of Systems Biology4 unknown5 Department of Acoustic Technology
Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.