Gee et al. used a yeast display to identify a peptide (“velcro”) that, when conjugated to a very low-affinity, TCR-specific peptide, enhanced the affinity of the binding peptide, converting it from a non-agonist to an agonist without altering the TCR/peptide:MHC interaction. Surprisingly, the velcro did not increase the peptide repertoire that bound a specific TCR, despite the increased affinity, indicating a conserved cross-reactivity profile of even weak affinity TCRs. This tool could be utilized to determine specificities of engineered TCRs, to discover new T cell agonists, and to increase the functional avidity of low-affinity peptides.
T cell receptors (TCRs) bind to peptide-major histocompatibility complex (pMHC) with low affinity (Kd approximately muM), which is generally assumed to facilitate cross-reactive TCR "scanning" of ligands. To understand the relationship between TCR/pMHC affinity and cross-reactivity, we sought to engineer an additional weak interaction, termed "velcro," between the TCR and pMHC to probe the specificities of TCRs at relatively low and high affinities. This additional interaction was generated through an eight-amino acid peptide library covalently linked to the N terminus of the MHC-bound peptide. Velcro was selected through an affinity-based isolation and was subsequently shown to enhance the cognate TCR/pMHC affinity in a peptide-dependent manner by approximately 10-fold. This was sufficient to convert a nonstimulatory ultra-low-affinity ligand into a stimulatory ligand. An X-ray crystallographic structure revealed how velcro interacts with the TCR. To probe TCR cross-reactivity, we screened TCRs against yeast-displayed pMHC libraries with and without velcro, and found that the peptide cross-reactivity profiles of low-affinity (Kd > 100 muM) and high-affinity (Kd approximately muM) TCR/pMHC interactions are remarkably similar. The conservation of recognition of the TCR for pMHC across affinities reveals the nature of low-affinity ligands for which there are important biological functions and has implications for understanding the specificities of affinity-matured TCRs.
Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Med
Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. Program in Immunology, Stanford University School of Medicine, Stanford, CA (2) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. Program in Immunology, Stanford University School of Medicine, Stanford, CA (3) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. Program in Immunology, Stanford University School of Medicine, Stanford, CA (4) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. (5) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. (6) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. (7) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. (8) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. Program in Immunology, Stanford University School of Medicine, Stanford, CA (9) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305; kcgarcia@stanford.edu. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305. Program in Immunology, Stanford University School of Medicine, Stanford, CA Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305.
