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Development of Photocrosslinkable Class I Peptide‐Major Histocompatibility Complex (pMHC) Monomers for Detecting Antigen‐Specific T Cells

A method using pMHC able to be crosslinked by UV irradiation acting on a 4‐azidosalicyclic acid (ASA) group on the peptide adjacent to the recognition sequences has been successful at labeling antigen‐specific T cells with excellent specificity and efficiency. However, studies with photocrosslinkable pMHC class I ligands are more challenging, since an ASA on the peptide interferes with T cell receptor (TCR) binding. In order to generate functional ligands to detect human TCRs, we first incorporated an ASA on the pMHC peptide through an 8‐residue extension. A second approach relied on designing a ligand where the ASA is attached to sites on the MHC molecule instead of the bound peptide, which allows for the incorporation of peptides in their native form. Seven recombinant human pMHC ligands were generated with, and then modified with ASA to produce photocrosslinkable HLA‐A*0201 molecules. Ultimately, flow cytometry will enable to determine which monomer is the most efficient at staining CMV‐specific CD8+ T cells in human peripheral blood mononuclear cells. In conclusion, we generated the precursors to a technology that allows staining of T cells specific for peptides bound to MHC class I molecules.