Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.     

The assembly of functional beta(2)-microglobulin-free MHC class I molecules that interact with peptides and CD8(+) T lymphocytes

Functional MHC class I molecules are expressed on the cell surface in the absence of beta(2)-microglobulin (beta(2)m) light chain that can interact with CD8(+) T lymphocytes. Whether their assembly requires peptide binding and whether their recognition by CD8(+) T lymphocytes involves the presentation of peptide epitopes remains unknown. We show that beta(2)m-free H-2D(b) assembles with short peptides that are approximately 9 amino acid residues in length, akin to ligands associated with completely assembled beta(2)m(+) H-2D(b). Remarkably, a subset of the peptides associated with the beta(2)m-free H-2D(b) has an altered anchor motif. However, they also include peptides that contain a beta(2)m(+)H-2D(b) binding anchor motif. Further, the H-2K(b)- and H-2D(b)-restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2(b) class I in beta(2)m-deficient cells and are recognized by epitope-specific CD8(+) T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of beta(2)m with peptides and form CD8(+) T lymphocyte epitopes.     

A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) presentation of gp33 revealed by comparative crystal structure.Analyses

LCMV infection of H-2(b) mice generates a CD8(+) CTL response mainly directed toward three immunodominant epitopes. One of these, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. The virus can escape immune recognition in the context of both these MHC class I molecules through single mutations of the peptide. In order to understand the underlying structural mechanism, we determined the crystal structures of both complexes. The structures reveal that the peptide is presented in two diametrically opposed manners by H-2D(b) and H-2K(b), with residues used as anchor positions in one MHC class I molecule interacting with the TCR in the other. Importantly, the peptide's N-terminal residue p1K protrudes from the binding cleft in H-2K(b). We present structural evidence that explains the functional consequences of single mutations found in escape variants.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Exact prediction of a natural T cell epitope.

T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351:290). These allele-specific motifs should allow exact prediction of natural T cell epitopes. H-2Kb-restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb-restricted epitope of ovalbumin, thought to be contained in the 19-mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible.     

Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)‐B*0702 and HLA‐B*0801 alleles in TB10.4 CD8+ T‐cell responses

The molecular definition of major histocompatibility complex (MHC) class I‐presented CD8⁺ T‐cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T‐cell‐based diagnostics of tuberculosis (TB) and the measurement of TB vaccine‐take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)‐A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I‐binding peptides from overlapping 9‐mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I‐binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N‐ and C‐termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off‐rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8⁺ T‐cell interaction with their nominal MHC class I‐peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8⁺ T cells from patients with active pulmonary TB. HLA‐B alleles served as the dominant MHC class I restricting molecules for anti‐Mtb TB10.4‐specific CD8⁺ T‐cell responses measured in CD8⁺ T cells from patients with pulmonary TB.     

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes

CD8⁺ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide / TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-γ and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-γ and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be usefol for inducing T lymphocytes with qualitatively different effector functions.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Estimating design space available for polyepitopes through consideration of major histocompatibility complex binding motifs

Major histocompatibility complex (MHC ) epitope presentation is needed for robust adaptive immune responses. Core peptide binding motifs for class I and class II MHC are 8–10 amino acids long, containing two or more “anchor” residues. These binding motifs define epitope anchor amino acid content and spacing, and knowledge of them has facilitated emergence of polyepitope vaccines. However, polyepitopes can exhibit “junctional epitopes” (neoepitopes interfering with vaccine function) resulting from juxtaposition of authentic epitopes. We have developed an algorithm for consideration of polyepitope sequence in light of MHC motifs to exhaustively identify all junctional-free polyepitope designs for any given set of authentic epitopes, and in so doing discovered that the number of such variants of any given polyepitope can be astronomically high. Our approach designs polyepitopes of any length, considers multiple MHC class I or class II motifs simultaneously and can be adapted to design variants of existing proteins with pre-selected epitope contents. We have also implemented the algorithm as a computer-based tool (CANVAC II), which we make available to interested parties. The vast diversity of junctional-free polyepitopes suggests that the number of potential T-helper epitope free protein variants may also be large, which may have implications for discovery of bioactive but non-immunogenic therapeutics.     

Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules

Naturally processed peptides presented by class I major histocompatibility complex (MHC) molecules display a characteristic allele specific motif of two or more essential amino acid side chains, the so-called peptide anchor residues, in the context of an 8-10 amino acid long peptide. Knowledge of the peptide binding motif of individual class I MHC molecules permits the selection of potential peptide antigens from proteins of infectious organisms that could induce protective T-cell-mediated immunity. Several methods have been developed for the prediction of potential class I MHC binding peptides. One is based on a simple scanning for the presence of primary peptide anchor residues in the sequence of interest. A more sophisticated technology is the utilization of predictive computer algorithms. Here, we have analyzed the experimental binding of 84 peptides selected on the basis of the presence of peptide binding motifs for individual class I MHC molecules. The actual binding was compared with the results obtained when analyzing the same peptides by two well-known, publicly available computer algorithms. We conclude that there is no strong correlation between actual and predicted binding when using predictive computer algorithms. Furthermore, we found a high number of false-negatives when using a predictive algorithm compared to simple scanning for the presence of primary anchor residues. We conclude that the peptide binding assay remains an important step in the identification of cytotoxic T lymphocyte (CTL) epitopes which can not be substituted by predictive algorithms.     

A pattern search method for putative anchor residues in T cell epitopes

The binding affinity between an antigenic peptide and its particular major histocompatibility complex (MHC) molecule seems to be largely determined by only a few residues. These residues have been called “anchors” because of their property of fitting into “pockets” inside the groove of the MHC molecule. To predict natural antigenic epitopes within a longer sequence, it therefore appears to be important to know the motif or pattern describing the anchors, i.e. the anchors amino acid residue preference and the distance between anchor residues. A large set of MHC class I-restricted peptides has been described. Peptide sequences vary in length and lack an obvious common sequence motif. For a list of peptides belonging to one type of MHC class I molecule, we describe a method to find the most prominent sequence motif with at least two anchor residues. Briefly, antigenic sequences are aligned, and two anchor positions are searched for, where all anchor residues share a high similarity. The alignments are scored according to the similarity of their anchor residues. We show that the motifs predicted for the MHC alleles A2.1, B27, K^b, K^d, D^b are in substantial agreement with experimental data. We derive binding motifs for the MHC class I alleles HLA-A1, All, B8, B14, H-2L^d and for the MHC class II alleles I-A^b and I-A^s. In some cases, higher scores were obtained by allowing a slight variation in the number of residues between anchors. Therefore, we support the view that the length of epitopes belonging to a particular class I MHC is not uniform. This method can be used to predict the natural short epitope inside longer antigenic peptides and to predict the epitopes anchor residues. Anchor motifs can be used to search for antigenic regions in sequences of infectious viruses, bacteria and parasites.     

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.     

Bovine lymphocyte antigen-A11--specific peptide motif as a means to identify cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1.

Major histocompatibility complex (MHC) class I molecules present 8- to 10-mer viral peptides to antiviral cytotoxic T lymphocytes (CTLs). Identification of the allele-specific peptide motifs (ASPMs) of class I molecules enables the prediction of potential CTL epitopes of a virus from its protein sequences. Based on the bovine herpesvirus 1 (BHV-1) protein sequences that conform to the BoLA-A11 ASPM that we identified previously, potential CTL epitopes of BHV-1 were synthesized for use in cytotoxicity assays with CTLs from BHV-1-immunized calves. A peptide binding assay used to select the peptides that are most likely to be CTL epitopes categorized the peptides into groups of high, intermediate, and low binding capacity. Synthetic peptides stimulated lymphocytes from BHV-1-immunized calves to secrete interferon-gamma. Groups of peptides from the major glycoproteins of BHV-1 restimulated CTLs in vitro and sensitized targets for lysis by means of restimulated bulk CTLs.     

Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue

Summary:  The epithelium of the human small intestine contains a large population of intraepithelial cytolytic αβ T-cell receptor (TCR) CD8αβ T lymphocytes (IE-CTLs), whose main role is to sustain epithelial integrity by rapidly eliminating infected and damaged cells. In mouse, the recognition of inducible/modified self-molecules, i.e. non-classical major histocompatibility complex (MHC) class I molecules, is mediated by the TCR and natural killer receptors (NKRs) co-expressed on the cell surface of a non-conventional autoreactive CD8αααβTCR cell subset. In contrast, in humans, the recognition of non-classical MHC class I molecules induced by stress and inflammation on intestinal epithelial cells (IECs) is principally mediated by NKRs expressed on conventional CD8αβαβTCR cells. By sensing microenvironmental signals of inflammation and stress through NKRs, IE-CTLs fine tune their TCR activation threshold. Furthermore, IE-CTLs under particular conditions, involving interleukin-15 upregulation, acquire the capacity to kill distressed intestinal epithelial cells in an antigen non-specific manner. Adaptive IE-CTLs appear hence to have autoreactive properties and modulate their immune response based on innate signals, reflecting the fitness of the tissue.     

Hb(64-76) epitope binds in different registers and lengths to I-Ek and I-Ak

The nature of peptide binding to MHC molecules is intrinsically degenerate, in what, one given MHC molecule can accommodate numerous peptides which are structurally diverse, and one given peptide can bind to different alleles. The structure of the MHC class II molecules allows peptides to extend out of the binding groove at both ends and these residues can potentially influence the stability and persistence of peptide/class II complexes. We have previously shown that both I-E(k) and I-A(k)-restricted T cell hybridomas could be generated against the Hb(64-76) epitope. In this study, we characterized the binding register of the Hb(64-76) epitope to I-A(k), and showed that it was shifted by one residue in comparison to its binding to I-E(k), and did not use a dominant anchor residue at P1. This conclusion was further supported by the modeling of the Hb(64-76) epitope bound to I-A(k), which revealed that all of its putative anchor residues fit into their corresponding pockets. We identified the naturally processed Hb epitopes presented by both I-E(k) and I-A(k), and found that they consisted of different species. Those associated with I-A(k) being 20-22 residues long, whereas, those found to I-E(k) contained 14-16 residues. These findings suggested that the lack of a dominant P1 anchor could be compensated by the selection of longer peptides. Overall, these studies revealed the Hb(64-76) epitope bound to I-E(k) and I-A(k) in distinct registers and lengths, demonstrating the plasticity MHC molecules have in generating distinct TCR ligands from the same amino acid sequence.     

Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity

Stable presentation of peptide epitope by major histocompatibility complex (MHC) class I molecules is a prerequisite for the efficient expansion of CD8(+) T cells. The construction of single-chain MHC class I molecules in which the peptide, β(2)-microglobulin, and MHC heavy chain are all joined together via flexible linkers increases peptide-MHC stability. We have expressed two T cell epitopes that may be useful in leukemia treatment as single-chain MHC class I molecules, aiming to develop a system for the expansion of antigen-specific CD8(+) T cells in vitro. Disulfide trap versions of these single-chain MHC molecules were also created to improve anchoring of the peptides in the MHC molecule. Unexpectedly, we observed that soluble disulfide trap single-chain molecules expressed in eukaryotic cells were prone to homodimerization, depending on the binding affinity of the peptide epitope. The dimers were remarkably stable and efficiently recognized by conformation-specific antibodies, suggesting that they consisted of largely correctly folded molecules. However, dimerization was not observed when the disulfide trap molecules were expressed as full-length, transmembrane-anchored molecules. Our results further emphasize the importance of peptide binding affinity for the efficient folding of MHC class I molecules.     

An assay for peptide binding to HLA-Cw*0102

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) molecules is based on the ability of peptides to stabilize MHC class I molecules synthesized by transporter associated with antigen processing (TAP)-deficient cell. The TAP-deficient cell line T2 has previously been used in the assembly assay to analyze peptide binding to HLA-A*0201 and -B*5101. In this study, we have extended this technique to assay for peptides binding to endogenous HLA-Cw*0102 molecules. We have analyzed the peptide binding of 20 peptides with primary anchor motifs for HLA-Cw*0102. One-third of the peptides analyzed bound with high affinity, half of the peptides examined did not bind, whereas the remaining peptides displayed intermediate binding activity. Interest in HLA-C molecules has increased significantly in recent years, since it has been shown that HLA-C molecules both can present peptides to cytotoxic T lymphocytes (CTL) and in addition are able to inhibit natural killer (NK)-mediated lysis.     

Nef-mediated MHC class I down-regulation unmasks clonal differences in virus suppression by SIV-specific CD8 T cells independent of IFN-γ and CD107a responses

CD8⁺ T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8⁺ T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4⁺ T cells. We used a set of SIV_mac239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I^high or MHC class I^intermediate. However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I^low) despite the observations that all CTL clones showed similar IFN-γ responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.     

Rational optimization of tumor epitopes using in silico analysis‐assisted substitution of TCR contact residues

Altered peptide ligands with increased affinity of the peptide–MHC complex for the TCR provide an alternative strategy to natural T‐cell epitopes for cancer immunotherapy, as they can recruit and stimulate stronger T‐cell repertoires. However, it remains unclear how alterations of the TCR contact residues improve the interaction between the peptide–MHC complex and the TCR molecule. In this study, we introduced a molecular simulation strategy to optimize a tumor immunodominant epitope NY–ESO‐1_157–165 by the substitution of the potential TCR contact residues. We correlated molecule simulation with T‐cell activation capacity assay and detected the effect of modifications of TCR contact residues on T‐cell recognition. An agonist peptide W5F with substitution at Trp5 with Phe was identified and it exhibits a stronger ability to induce a cross‐reactive CTL response with the WT peptide. Additionally, the W5F‐induced CTL could be maintained with the WT peptide and possess higher capacity in lysing native NY–ESO‐1‐expressing tumor cells. These results provide important insights into the enhanced immunogenicity of epitopes through substitution at the TCR contact sites and revealed a novel molecular simulation approach for rational therapeutic peptide vaccine design.