Plasticity in the contribution of T cell receptor variable region residues to binding of peptide-HLA-A2 complexes

Abstract

One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this "germline codon" hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs.

Keywords: CDR; MHC; SOE; SPR; T cell receptor; T cell receptors; TCR; complementarity-determining region; directed evolution; germline codon bias; major histocompatibility complex; single-chain; splicing by overlap extension; surface plasmon resonance; yeast display.

Figure 1. Yeast display and isolation of INR1-T1 (T1) single-chain TCR variable fragments (scTv)

(a) Schematic of scTv fusions for the human TCR, T1, which recognizes MART-1 peptides in presented in the context of HLA-A2. (b) The T1 TCR was cloned as a scTv and expressed on the surface of yeast (left panels). Yeast surface of scTV fusions were monitored for expression of an N-terminal tag [hemagluttanin (HA), black line] with anti-HA antibody and goat anti-mouse IgG alexa 647 secondary antibody or secondary only as a control (gray). The negative peak is is due to yeast that have lost plasmid, and serves as an internal control for each induced yeast sample. Cells were incubated with anti-Vβ16 and anti-Vα2 antibodies followed by goat anti-mouse IgG alexa 647 secondary antibody, or secondary only as a control (gray). Cells were incubated with MART-1 peptide (ELAGIGILT)/HLA-A2 dimer at 100 nM. A random mutagenesis library was generated using the T1 scTv as a template and sorted with anti-Vβ16 antibody and goat anti-mouse IgG alexa 647 secondary antibody. Clone T1-S18 was isolated after two rounds of sorting (middle panels). Site directed libraries were made in CDR3 loop regions of the stabilized T1-S18 clone. The high affinity clone, T1-S18.45, was isolated following 4 rounds of sorting with MART-1 peptide (ELAGIGILT)/HLA-A2 dimer (right panels, black line). Histograms are representative of 3 or more experiments.

Figure 2. Purity and surface plasmon resonance of soluble MART-1-specific, single-chain TCR T1 and its engineered variants

T1 wt, T1-S18, and T1-S18.45 were expressed in the E.coli pET28 expression system, refolded from inclusion bodies, and purified by Ni-column and size exclusion chromatography. (a) SDS-PAGE of purified scTvs and molecular weight markers (M). (b) SPR trace of MART-1 (ELAGIGILTV)/HLA-A2 binding immobilized T1-S18.45. Fitted parameters (K_D, k_on, k_off) are shown in the inset. (c) Table showing the binding affinities of the T1-derived TCRs for variants of the MART-1 peptide.

Figure 3. Binding of peptide-loaded antigen presenting cells by soluble T1-S18.45 scTv

(a) Titration of biotinylated T1-S18.45 scTv on antigen-presenting cell line T2 (HLA-A2+) preloaded with MART-1 peptide (1 µM) or null peptide, SL9 (1 µM). Cells were stained with 3.9 nM (tan), 7.8 nM (gray), 15.2 nM (red), 31.1 nM (purple), 62.5 nM (green), 250 nM (blue), or 1 µM (black) biotinylated T1-S18.45 scTv, followed by SA:PE. Data shown is representative of 4 experiments. (b) Mean fluorescence unit (MFU) values of histograms in (a) are plotted versus scTv-biotin concentration.

Figure 4. Crystal structures of Vα2-containing TCRs showing MHC contact positions in CDR1α and CDR2α loops

CDR1α residues D27, R28, and Q31 (a,c,e), and CDR2α residues Y51 and S52 (b,d,f) assessed in the alanine scanning study are highlighted in the structures of the A6 (a,b), Mel5 (c,d), and DMF5 (e,f) TCRs, which all contain the Vα2 region. PDB files 1AO7, 3HG1, and 3QDG were used, respectively, in PyMol. TCR residue positions are indicated in black. MHC residues are italicized in gray.

Figure 5. Alanine scanning mutagenesis of Vα2-containing single-chain TCRs

Binding titrations of yeast-displayed mutants of Vα2-containing single-chain TCR fragments (scTv): (a), A6-X15 specific for Tax/HLA-A2, (b) 868 Z11 specific for SL9/HLA-A2, and (c) T1-S18.45 specific for MART-1/HLA-A2. Alanine mutants were stained in triplicate with 8 nM, 40 nM, 200 nM, 1 µM, and 5 µM cognate peptide/HLA-A2 monomers followed by PE-conjugated streptavidin. Normalized percent max mean fluorescence intensity (MFI) is plotted against cognate peptide/HLA-A2 monomer concentration. Error bars represent standard deviations of triplicate experiments. (d) Fold changes in binding were determined by the scTv concentrations at one-half max wild-type binding from titrations in Figure 4a,b,c. Error bars represent standard deviations of triplicate experiments.

Figure 6. In vitro selected mutants of yeast-display libraries of the MART-1-specific TCR (T1-S18.45)

Yeast display libraries at position 31 in CDR1α (a) and position 51 in CDR2α (b) were generated, stained with 45 nM MART-1 (ELAGIGITV)/HLA-A2, and selected by fluorescence activated cell sorting. The top staining (8–10%) clones were isolated, and amino acid distribution was determined by 454 sequencing. Amino acid residues that were positively and negative selected are indicated on the y-axis as a function of the ratio of the logarithm of amino acid frequency post-selection divided by the logarithm of the frequency pre-selection. A total of 11,736 and 13,600 clones from the unselected position 31 library and selected position 31 library were sequenced, respectively; a total of 6,806, and 24,532 from the unselected position 51 library and selected position 51 library were sequenced, respectively.