Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 52, Pages 19033-19038Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0507554102
Keywords
antigen specificity; major histocompatibility complex; T cell activity
Categories
Funding
- NIAID NIH HHS [AI61173, R01 AI024157, R56 AI061173, R01 AI061173, AI24157, R37 AI024157] Funding Source: Medline
- NIGMS NIH HHS [R01 GM055767, GM55767] Funding Source: Medline
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The T cell receptor (TCR) alpha beta 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 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.
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