Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function

K. Scott Weber, David L. Donermeyer, Paul M. Allen, David M. Kranz

Research output: Contribution to journalArticle

Abstract

The T cell receptor (TCR) αβ 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-Ek), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (Vβ-linker-Vα) 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-Ek 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.

Original languageEnglish (US)
Pages (from-to)19033-19038
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number52
DOIs
StatePublished - Dec 27 2005

Keywords

  • Antigen specificity
  • Major histocompatibility complex
  • T cell activity

ASJC Scopus subject areas

  • General

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