Class II-Restricted T Cell Receptor Engineered in vitro for Higher Affinity Retains Peptide Specificity and Function

• Published in: Proceedings of the National Academy of Sciences - PNAS 2005-12, Vol.102 (52), p.19033-19038
• Main Authors: K. Scott Weber, David L. Donermeyer, Allen, Paul M., Kranz, David M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antigens; Biological Sciences; Dimerization; Dimers; Flow Cytometry; Gene Library; Genes, MHC Class II; Genetic mutation; Half lives; Hot Temperature; Hybridomas; Immunology; Kinetics; Libraries; Ligands; Lymphocyte Activation; Molecular Sequence Data; Mutation; Peptides; Peptides - chemistry; Protein Binding; Protein Engineering - methods; Receptors; Receptors, Antigen, T-Cell - metabolism; Receptors, Antigen, T-Cell, alpha-beta - chemistry; Receptors, Antigen, T-Cell, alpha-beta - physiology; Surface Plasmon Resonance; T cell antigen receptors; T cell receptors; T lymphocytes; T-Lymphocytes - cytology; T-Lymphocytes - metabolism; Time Factors; Transfection; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0507554102

The T cell receptor (TCR) α β heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin$(Hb/I-E^k)$to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR$(V\beta-linker-V\alpha)$was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with$(Hb/I-E^k)$to isolate a panel of affinity mutants with KDvalues as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation.