Brian M. Baker

The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site.

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 Å) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, ΔTm = 16°C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a “closed conformation” of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes.

PREDICTION OF BINDING ENERGETICS FROM STRUCTURE USING EMPIRICAL PARAMETERIZATION

We have presented an empirical method that can be used to predict the binding energetics for protein-protein or protein-peptide interactions from three-dimensional structures. The approach differs from other empirical methods in yielding a thermodynamic description of the binding process, including delta Cp, delta H degree, and delta S degree, rather than predicting delta G degree alone. These thermodynamic terms can provide a wealth of detail about the nature of the interaction, and, if sufficient experimental data are available for comparison, a greater assessment of the accuracy of the calculations. A recurring theme throughout this article is the need for more complete thermodynamic and structural characterizations of protein-ligand interactions. This includes not only characterization of the binding delta H degree, delta S degree, and delta Cp, but a thorough investigation into equilibria linked to binding, such as protonation, ion binding, and conformational changes. Sufficient data will allow parameterization on binding data rather than protein unfolding data. Further inclusion of information obtained from unfolding studies is not likely to generate significant improvement in the accuracy of the calculations. As additional binding data become available, the parameterization can be further extended to include relationships derived from analyses of these data. Not only will this increase accuracy and thus confidence, but allow extension of the method of additional types of interactions.

Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity

Srivastava et al. define a new and improved way to predict immunoprotective cancer neoepitopes based in part on the difference in MHC-binding scores between the mutant epitope and its wild-type counterpart. Remarkably, all neoepitopes that elicited tumor regression bound to class I MHC molecules with very low affinity.

Poor immunogenicity of a self/tumor antigen derives from peptide–MHC-I instability and is independent of tolerance

Understanding the mechanisms underlying the poor immunogenicity of human self/tumor antigens is challenging because of experimental limitations in humans. Here, we developed a human-mouse chimeric model that allows us to investigate the roles of the frequency and self-reactivity of antigen-specific T cells in determination of the immunogenicity of an epitope (amino acids 209-217) derived from a human melanoma antigen, gp100. In these transgenic mice, CD8+ T cells express the variable regions of a human T cell receptor (hTCR) specific for an HLA-A*0201-restricted gp100(209-217). Immunization of hTCR-transgenic mice with gp100(209-217) peptide elicited minimal T cell responses, even in mice in which the epitope was knocked out. Conversely, a modified epitope, gp100(209-217(2M)), was significantly more immunogenic. Both biological and physical assays revealed a fast rate of dissociation of the native peptide from the HLA-A*0201 molecule and a considerably slower rate of dissociation of the modified peptide. In vivo, the time allowed for dissociation of peptide-MHC complexes on APCs prior to their exposure to T cells significantly affected the induction of immune responses. These findings indicate that the poor immunogenicity of some self/tumor antigens is due to the instability of the peptide-MHC complex rather than to the continual deletion or tolerization of self-reactive T cells.

Conformational changes and flexibility in T-cell receptor recognition of peptide-MHC complexes

A necessary feature of the immune system, TCR (T-cell receptor) cross-reactivity has been implicated in numerous autoimmune pathologies and is an underlying cause of transplant rejection. Early studies of the interactions of αβ TCRs (T-cell receptors) with their peptide–MHC ligands suggested that conformational plasticity in the TCR CDR (complementarity determining region) loops is a dominant contributor to T-cell cross-reactivity. Since these initial studies, the database of TCRs whose structures have been solved both bound and free is now large enough to permit general conclusions to be drawn about the extent of TCR plasticity and the types and locations of motion that occur. In the present paper, we review the conformational differences between free and bound TCRs, quantifying the structural changes that occur and discussing their possible roles in specificity and cross-reactivity. We show that, rather than undergoing major structural alterations or ‘folding’ upon binding, the majority of TCR CDR loops shift by relatively small amounts. The structural changes that do occur are dominated by hinge-bending motions, with loop remodelling usually occurring near loop apexes. As predicted from previous studies, the largest changes are in the hypervariable CDR3α and CDR3β loops, although in some cases the germline-encoded CDR1α and CDR2α loops shift in magnitudes that approximate those of the CDR3 loops. Intriguingly, the smallest shifts are in the germline-encoded loops of the β-chain, consistent with recent suggestions that the TCR β domain may drive ligand recognition.

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting.

Background: Current peptide vaccines may select suboptimal antigen-specific T-cells from polyclonal populations. Results: A combinatorial peptide library screen was used to generate an optimal ligand that could preferentially activate a known effective T-cell clonotype. Conclusion: Rationally designed altered peptide ligands may enable the preferential selection of high quality, antigen-sensitive T-cell clonotypes. Significance: This proof-of-principle study could facilitate the development of more effective peptide vaccination strategies. Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8+ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8+ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8+ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201+ individuals that were capable of efficient HLA A*0201+ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.

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

The use of “anchor-fixed” altered peptide ligands is of considerable interest in the development of therapeutic vaccines for cancer and infectious diseases, but the mechanism by which successful altered peptide ligands elicit enhanced immunity is unclear. In this study, we have determined the crystallographic structure of a major tumor rejection Ag, gp100209–217, in complex with the HLA-A*0201 (HLA-A2) molecule, as well as the structure of a modified version of the peptide which substitutes methionine for threonine at position 2 (T2M; gp100209–2M). The T2M-modified peptide, which is more immunogenic in vitro and in vivo, binds HLA-A2 with a ∼9-fold greater affinity and has a ∼7-fold slower dissociation rate at physiological temperature. Within the limit of the crystallographic data, the T2M substitution does not alter the structure of the peptide/HLA-A2 complex. Consistent with this finding, in peripheral blood from 95 human subjects, we were unable to identify higher frequencies of T cells specific for either the native or modified peptide. These data strongly support the conclusion that the greater immunogenicity of the gp100209–2M peptide is due to the enhanced stability of the peptide/MHC complex, validating the anchor-fixing approach for generating therapeutic vaccine candidates. Thermodynamic data suggest that the enhanced stability of the T2M-modified peptide/HLA-A2 complex is attributable to the increased hydrophobicity of the modified peptide, but the gain due to hydrophobicity is offset considerably by the loss of a hydrogen bond made by the native peptide to the HLA-A2 molecule. Our findings have broad implications for the optimization of current vaccine-design strategies.

TCRs Used in Cancer Gene Therapy Cross-React with MART-1/Melan-A Tumor Antigens via Distinct Mechanisms

T cells engineered to express TCRs specific for tumor Ags can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Although DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity toward both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same Ags and the finding that TCR-binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.

Cutting Edge: Evidence for a Dynamically Driven T Cell Signaling Mechanism

T cells use the αβ TCR to bind peptides presented by MHC proteins (pMHC) on APCs. Formation of a TCR–pMHC complex initiates T cell signaling via a poorly understood process, potentially involving changes in oligomeric state, altered interactions with CD3 subunits, and mechanical stress. These mechanisms could be facilitated by binding-induced changes in the TCR, but the nature and extent of any such alterations are unclear. Using hydrogen/deuterium exchange, we demonstrate that ligation globally rigidifies the TCR, which via entropic and packing effects will promote associations with neighboring proteins and enhance the stability of existing complexes. TCR regions implicated in lateral associations and signaling are particularly affected. Computational modeling demonstrated a high degree of dynamic coupling between the TCR constant and variable domains that is dampened upon ligation. These results raise the possibility that TCR triggering could involve a dynamically driven, allosteric mechanism.

Structural and dynamic control of T-cell receptor specificity, cross-reactivity, and binding mechanism.

Over the past two decades, structural biology has shown how T‐cell receptors engage peptide/major histocompatibility complex (MHC) complexes and provided insight into the mechanisms underlying antigen specificity and cross‐reactivity. Here we review and contextualize our contributions, which have emphasized the influence of structural changes and molecular flexibility. A repeated observation is the presence of conformational melding, in which the T‐cell receptor (TCR), peptide, and in some cases, MHC protein cooperatively adjust in order for recognition to proceed. The structural changes reflect the intrinsic dynamics of the unligated proteins. Characterization of the dynamics of unligated TCR shows how binding loop motion can influence TCR cross‐reactivity as well as specificity towards peptide and MHC. Examination of peptide dynamics indicates not only peptide‐specific variation but also a peptide dependence to MHC flexibility. This latter point emphasizes that the TCR engages a composite peptide/MHC surface and that physically the receptor makes little distinction between the peptide and MHC. Much additional evidence for this can be found within the database of available structures, including our observations of a peptide dependence to the TCR binding mode and structural compensations for altered interatomic interactions, in which lost TCR‐peptide interactions are replaced with TCR‐MHC interactions. The lack of a hard‐coded physical distinction between peptide and MHC has implications not only for specificity and cross‐reactivity but also the mechanisms underlying MHC restriction as well as attempts to modulate and control TCR recognition.