Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition

Abstract

Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg 65 and Lys 66 and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg 65 -Lys 66 region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.Recognition of a peptide bound to a major histocompatibility complex protein (peptide-MHC) 1 by the ␣␤ T cell receptor (TCR) is necessary for the initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. TCRs bind peptide-MHC in a diagonal-to-orthogonal fashion, interacting with elements of both the peptide and the MHC (1-3). Numerous studies have probed the interfaces between TCRs and their ligands through the use of altered peptides or site-directed mutagenesis of the MHC (e.g. see . These studies have been useful in identifying hot spots within individual interfaces, as well as predicting the biophysical mechanisms by which T cell receptors bind their ligand (9).In our studies of TCR recognition of the human class I MHC HLA-A*0201 (referred to as HLA-A2) presenting the Tax peptide (sequence LLFGYPVYV; see Ref. 10), we identified a functional hot spot consisting of arginine 65 and lysine 66 on the HLA-A2 ␣1 helix (4). In a mutagenesis experiment involving 15 HLA-A2 amino acids contacted by the Tax-HLA-A2-specific ␣␤ T cell receptor A6, only two mutations, Arg 65 3 Ala (R65A) and Lys 66 3 Ala (K66A), resulted in significantly reduced T cell effector functions when the mutants were used to present the Tax peptide to T cells expressing the A6 receptor. Similar results were found with T cells expressing a different Tax-HLA-A2-specific TCR, B7. Lys 66 was also identified as a critical position influencing T cell reactivity in at least one other mutagenesis study of HLA-A2 (8).The general nature of the Arg 65 -Lys 66 hot spot in the recognition of Tax...

Increased Immunogenicity of an Anchor-Modified Tumor-Associated Antigen Is Due to the Enhanced Stability of the Peptide/MHC Complex: Implications for Vaccine Design

“…Consistent with similar quantitative analyses of peptide dissociation from class I MHC molecules (23,(41)(42)(43), the majority of the data in Fig. 6 were best fit with a double-exponential decay function, consisting of a slow phase accounting for the bulk of the data and a minor, faster phase typically accounting for much smaller (5-10%) of the total amplitude.…”

“…6 were best fit with a double-exponential decay function, consisting of a slow phase accounting for the bulk of the data and a minor, faster phase typically accounting for much smaller (5-10%) of the total amplitude. In similar measurements, the minor phase has been attributed to the presence of a small amount of peptide/H chain heterodimer, from which peptide more rapidly dissociates (23,41). The major phase thus represents the more physiologically relevant dissociation of peptide from the complete class I MHC complex.…”

Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility

“…anisotropy, using peptides fluorescently labeled at position 7 (21,22). The labeled ALG peptide had a substantially slower rate of dissociation than the labeled AAG peptide, ranging from 10-fold slower at 15°C to 45-fold slower at 37°C (the initial anisotropy of the ALG complex was 10 -20% lower at every temperature, consistent with the greater flexibility of the peptide) (Fig.…”

“…Peptide Dissociation Kinetics-Peptide dissociation from HLA-A2 was monitored using fluorescence anisotropy as described previously (21,22), utilizing a temperature-controlled Beacon 2000 instrument to monitor steady-state anisotropy (Invitrogen). Solution conditions were 10 mM HEPES, 150 mM NaCl, pH 7.4.…”

“…6a). We next used an Eyring analysis of dissociation rates measured from 15 to 37°C to determine the thermodynamics of dissociation (21,22). As shown in Fig.…”