title: Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function.
abstract: The T cell receptor (TCR) alphabeta 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 (Vbeta-linker-Valpha) 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 K(D) values 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.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: K Scott
authors.initials: KS
authors.lastname: Weber
authors.forename: David L
authors.initials: DL
authors.lastname: Donermeyer
authors.forename: Paul M
authors.initials: PM
authors.lastname: Allen
authors.forename: David M
authors.initials: DM
authors.lastname: Kranz
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D005434:Flow Cytometry; D015723:Gene Library; D005802:Genes, MHC Class II; D006358:Hot Temperature; D006825:Hybridomas; D007700:Kinetics; D008024:Ligands; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000502:physiology*; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D008024:Ligands; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta