Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Abstract

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

FIGURE 1.

Anchor modification of the MART-1_27–35 peptide strengthens peptide binding to MHC but weakens TCR binding to pMHC. a, thermal stability monitored by circular dichroism indicates that the apparent T_m of the ALG·HLA-A2 complex is 14° higher than that of the AAG·HLA-A2 complex, consistent with the reported 40-fold increase in peptide binding affinity (2). b, although it recognizes the AAG·HLA-A2 and EAA·HLA-A2 complexes with moderate affinities, the DMF5 TCR binds the ALG·HLA-A2 complex very weakly, consistent with the loss in antigenicity seen with the ALG peptide.

FIGURE 2.

The center of the ALG but not the AAG peptide shows structural heterogeneity in the HLA-A2 peptide binding groove. a, superimposition of the peptides in both asymmetric units from the new and previously solved AAG·HLA-A2 structures. The color scheme is at the bottom of the figure and maintained in each panel. b, 2F_o − F_c simulated annealing OMIT maps of electron density contoured at 1 σ for the peptides in the asymmetric unit of the new AAG·HLA-A2 structure. The glycine at position 5 is highlighted in green. c, structural comparisons of the centers of the AAG and ALG peptides from the available structures. Peptides are rotated ∼45° toward the viewer relative to their orientation in panels a and b. The panel at the top shows each peptide superimposed, including both molecules in each asymmetric unit. The glycine at position 5 is circled. The bottom left panel shows the various AAG peptides, revealing a spread in the position of the Gly-5 α carbon, but no flip in the backbone. The bottom right panel shows the ALG peptides, revealing not only a greater spread in the position of the Gly-5 α carbon, but also the flip in the backbone at the Gly-5 amide nitrogen and Ile-4 carbonyl oxygen.

FIGURE 3.

NMR identifies major and minor conformations for the ALG peptide but only a single, overlapping conformation for the AAG peptide when bound to HLA-A2. The figure shows superimposed ¹H-¹⁵N heteronuclear single quantum correlation spectra for the AAG·HLA-A2 and ALG·HLA-A2 complexes with the Gly-5 amide nitrogen in each peptide ¹⁵N-labeled. Data were collected at 4 °C. A single cross-peak was observed in the AAG spectrum. Two cross-peaks (peak A and peak B, with the indicated distribution) were observed in the ALG spectrum. The major peak (peak A) in the ALG spectrum overlapped with the single peak seen in the AAG spectrum.

FIGURE 4.

Molecular dynamics simulations indicate greater flexibility for the ALG than the AAG peptide when bound to HLA-A2. a, ψ/φ distributions for Gly-5 and Ile-4 for the AAG simulation at 300 K. Only the crystallographically observed non-flipped conformation was observed. b, ψ/φ distributions for Gly-5 and Ile-4 for the ALG simulation at 300 K. In addition to the non-flipped conformation, the flipped conformation was populated at a level of 15%. This occurred due to a single conformational flip that persisted for ∼8 ns. c, increasing the simulation temperature to 330 K resulted in the AAG peptide populating the flipped conformation to 7%. d, increasing the simulation temperature to 330 K increased population of the ALG flipped conformation to 39%. e, B-factors calculated from the simulations indicate greater flexibility across the N-terminal half of the ALG peptide. f, the dominant non-flipped conformation of the ALG peptide is more dynamic than the flipped conformation.

FIGURE 5.

Anchor modification of the MART-1_27–35 peptide alters the flexibility of the HLA-A2 peptide binding groove. a, B-factors calculated from the AAG and ALG simulations at 300 K for the α carbons of the HLA-A2 α1 and α2 helices. Flexibility of the central portion of the α1 helix (outlined) is enhanced, whereas flexibility of the central portion of the α2 helix (also outlined) is decreased. b, differences in calculated B-factors between the ALG and AAG simulations mapped to the structure of the pMHC complex. Red indicates a gain in flexibility resulting from anchor modification, and blue indicates a loss in flexibility resulting from anchor modification. The regions corresponding to the centers of the α1 and α2 helices outlined in panel a are indicated. c, heat map of differences in pairwise distance fluctuations between the α carbons of the α1 and α2 helices between the ALG and AAG simulations. Red indicates greater fluctuations resulting from anchor modification. The region outlined with a dashed line highlights the greater fluctuations resulting from the increased mobility of the center of the α1 helix. The smaller region outlined with a solid line indicates the significantly enhanced distance fluctuations between the regions of the α1 and α2 helices immediately adjacent to the center of the peptide. d, averaged B-factors of atoms of the P2 side chains and those of His-70 and Tyr-99 in the AAG and ALG simulations at 300 K. The greater flexibilities in the ALG complex indicate a direct connection between dynamics at the position 2 anchor, the peptide backbone, and the center of the α1 helix. e, structural relationship between Leu-2 in the HLA-A2 P2 pocket and His-70 and Tyr-99 of the peptide binding groove. Red dashed lines show hydrogen bonds between Tyr-99 and His-70 and Gly-3 of the peptide.

FIGURE 6.

Peptide dissociation thermodynamics are consistent with a higher entropy for the ALG·HLA-A2 complex. a, peptide dissociation at 37 °C measured by fluorescence anisotropy. The much slower dissociation of the ALG peptide is readily apparent. b, Eyring analysis of peptide dissociation data from 15 to 37 °C. The activation thermodynamics indicate that the ALG·HLA-A2 complex peptide gains less entropy moving from the bound to the transition state, consistent with the greater flexibility seen in the ALG·HLA-A2 complex.