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.

Fig. 1.

Yeast display of 3.L2 scTCR and overview of engineering process. (A) Schematic of the 3.L2 scTCR (Vβ-linker-Vα), hemagluttinin (HA) tag, and c-myc tag expressed as an Aga-2 fusion protein on the surface of yeast. (B) Histogram overlay of yeast displayed scTCRs (WT and M2) stained with CAb. (C) Overview of 3.L2 engineering showing the selection process for each clone, SPR measured K_D, free energy of binding differences (kcal/mol), and the location of CDR affinity mutations (purple or green circles represent mutations in respective CDR regions: Vβ1-2-3 and Vα1-2-3).

Fig. 2.

Vα and Vβ sequences of 3.L2 TCR and mutants. TCR alignment shows 3.L2 WT amino acids and the mutations isolated during the selection for yeast surface display (blue) and affinity selection (red). The four 2C stabilizing mutations cloned into WT are highlighted (cyan).

Fig. 3.

Hb/I-E^k binding by 3.L2 scTCR mutants. (A) Histograms of yeast displayed 3.L2 scTCR clones stained with 2μM Hb/I-E^k-Ig dimer. (B and C) Sample SPR traces of immobilized Hb/I-E^k binding by soluble scTCR 3.L2 mutants, M1 (B) and M15 (C), at the indicated concentrations.

Fig. 4.

Hb/MCC and Hb/D73 peptide overlays and peptide-mediated IL-2 release from T cell transfected with WT 3.L2 TCR or high-affinity mutant TCRs. (A) Hb/MCC peptide overlay with Hb shown in red and MCC in blue and sequence alignment of Hb, MCC, and D73. Coordinates of Hb/I-E^k [Protein Data Bank (PDB) ID code 1FNG] and MCC/I-E^k (PDB ID code 1KT2) were used to compare the peptides by aligning the I-Eα chains using the insight ii suite of programs (Molecular Simulations, Waltham, MA). (B) IL-2 release measured in a biological indicator assay (cpm) at various concentrations of WT Hb (blue curves) or MCC (red curves) presented by APCs. (C)Hb/D73 peptide overlay with Hb shown in blue and D73 in green, using coordinates of Hb/I-E^k and D73/I-E^k (PDB ID code 1FNE) as described above. (D) IL-2 release measured at various concentrations of the WT Hb (blue curves) or D73 peptide (green curves) presented by APCs.