Generation of CTL recognizing an HLA-A*0201-restricted epitope shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 tumor antigens: implication in a broad-spectrum tumor immunotherapy

MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are expressed in a significant proportion of primary and metastatic tumors of various histological types and are targets of tumor Ag-specific CTL. Individual MAGE-A expression varies from one tumor type to the other but, overall, the large majority of tumors expresses at least one MAGE-A Ag. Therefore, targeting epitopes shared by all MAGE-A Ags would be of interest in immunotherapy against a broad spectrum of cancers. In the present study, we describe a heteroclitic MAGE-A peptide (p248V9) that induces CTL in vivo in HLA-A*0201 transgenic HHD mice and in vitro in healthy donors. These CTL are able to recognize two low HLA-A*0201 affinity peptides differing at their C-terminal position and derived from MAGE-A2, -A3, -A4, -A6, -A10, and -A12 (p248G9) and MAGE-A1 (p248D9). Interestingly, p248V9-specific CTL respond to endogenous MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 in an HLA-A*0201-restricted manner and recognize human HLA-A*0201(+)MAGE-A(+) tumor cells of various histological origin. Therefore, this heteroclitic peptide may be considered as a potent candidate for a broad-spectrum tumor vaccination.     

T cell responses to HLA-A*0201-restricted peptides derived from human alpha fetoprotein

alpha fetoprotein (AFP)-derived peptide epitopes can be recognized by human T cells in the context of MHC class I. We determined the identity of AFP-derived peptides, presented in the context of HLA-A*0201, that could be recognized by the human (h) T cell repertoire. We screened 74 peptides and identified 3 new AFP epitopes, hAFP(137-145), hAFP(158-166), and hAFP(325-334), in addition to the previously reported hAFP(542-550.) Each possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent class I binding assay. The peptides were stable for 2-4 h in an off-kinetics assay. Each peptide induced peptide-specific T cells in vitro from several normal HLA-A*0201 donors. Importantly, these hAFP peptide-specific T cells also were capable of recognizing HLA-A*0201(+)/AFP(+) tumor cells in both cytotoxicity assays and IFN-gamma enzyme-linked immunospot assays. The immunogenicity of each peptide was tested in vivo with HLA-A*0201/K(b)-transgenic mice. After immunization with each peptide emulsified in CFA, draining lymph node cells produced IFN-gamma on recognition of cells stably transfected with hAFP. Furthermore, AFP peptide-specific T cells could be identified in the spleens of mice immunized with dendritic cells transduced with an AFP-expressing adenovirus (AdVhAFP). Three of four AFP peptides could be identified by mass spectrometric analysis of surface peptides from an HLA-A*0201 human hepatocellular carcinoma (HCC) cell line. Thus, compelling immunological and physiochemical evidence is presented that at least four hAFP-derived epitopes are naturally processed and presented in the context of class I, are immunogenic, and represent potential targets for hepatocellular carcinoma immunotherapy.     

Investigation of peptide involvement in T cell allorecognition using recombinant HLA class I multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.     

Random screening of proteins for HLA-A*0201-binding nine-amino acid peptides is not sufficient for identifying CD8 T cell epitopes recognized in the context of HLA-A*0201

HLA-A2 is the most frequent HLA molecule in Caucasians with HLA-A*0201 representing the most frequent allele; it was also the first human HLA allele for which peptide binding prediction was developed. The Bioinformatics and Molecular Analysis Section of the National Institutes of Health (BIMAS) and the University of Tübingen (Syfpeithi) provide the most popular prediction algorithms of peptide/MHC interaction on the World Wide Web. To test these predictions, HLA-A*0201-binding nine-amino acid peptides were searched by both algorithms in 19 structural CMV proteins. According to Syfpeithi, the top 2% of predicted peptides should contain the naturally presented epitopes in 80% of predictions (www.syfpeithi.de). Because of the high number of predicted peptides, the analysis was limited to 10 randomly chosen proteins. The top 2% of peptides predicted by both algorithms were synthesized corresponding to 261 peptides in total. PBMC from 10 HLA-A*0201-positive and CMV-seropositive healthy blood donors were tested by ex vivo stimulation with all 261 peptides using crossover peptide pools. IFN-gamma production in T cells measured by CFC was used as readout. However, only one peptide was found to be stimulating in one single donor. As a result of this work, we report a potential new T cell target protein, one previously unknown CD8-T cell-stimulating peptide, and an extensive list of CMV-derived potentially strong HLA-A*0201-binding peptides that are not recognized by T cells of HLA-A*0201-positive CMV-seropositive donors. We conclude that MHC/peptide binding predictions are helpful for locating epitopes in known target proteins but not necessarily for screening epitopes in proteins not known to be T cell targets.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer

Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201-restricted peptides of MAGE-A3: 112-120 (KVAELVHFL) or MAGE-A3: 271-279 (FLWGPRALV), and T cell clones were generated. MAGE-A3-specific TCR α- and β-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112-120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti-MAGE-A3 TCR.     

Coupling in silico and in vitro analysis of peptide-MHC binding: a bioinformatic approach enabling prediction of superbinding peptides and anchorless epitopes

The ability to define and manipulate the interaction of peptides with MHC molecules has immense immunological utility, with applications in epitope identification, vaccine design, and immunomodulation. However, the methods currently available for prediction of peptide-MHC binding are far from ideal. We recently described the application of a bioinformatic prediction method based on quantitative structure-affinity relationship methods to peptide-MHC binding. In this study we demonstrate the predictivity and utility of this approach. We determined the binding affinities of a set of 90 nonamer peptides for the MHC class I allele HLA-A*0201 using an in-house, FACS-based, MHC stabilization assay, and from these data we derived an additive quantitative structure-affinity relationship model for peptide interaction with the HLA-A*0201 molecule. Using this model we then designed a series of high affinity HLA-A2-binding peptides. Experimental analysis revealed that all these peptides showed high binding affinities to the HLA-A*0201 molecule, significantly higher than the highest previously recorded. In addition, by the use of systematic substitution at principal anchor positions 2 and 9, we showed that high binding peptides are tolerant to a wide range of nonpreferred amino acids. Our results support a model in which the affinity of peptide binding to MHC is determined by the interactions of amino acids at multiple positions with the MHC molecule and may be enhanced by enthalpic cooperativity between these component interactions.     

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Identification and functional perspective of a novel HLA-A allele: A*0279

A novel HLA-A allele, HLA-A*0279, was identified using PCR-SSP and PCR-SBT methods. It is inheritable. HLA-A*0279 differs from HLA-A*020601 by a single nucleotide at position 497 in exon 3, leading to an amino acid change from Threonine to Isoleucine at the alpha2 helix of HLA molecule. To investigate whether the altered amino acid residue could affect its peptide-binding repertoire, we compared the predicted crystal structure of HLA-A*020601 and HLA-A*0279 by Swiss-PdbViewer software analysis. We found that the crystal structure of the two molecules is very similar except for a difference in the number of hydrogen bonds they can possibly form, which in turn could affect their structural stability. To test whether HLA-A*0279 has the ability to cross-present A*0201 - restricted peptides to T cells, the full lenght cDNA of HLA-A*0201, -A* 020601 and -A*0279 were respectively transfected into COS-7 cells, which were then used as targets in IFN-gamma release Elispot assay. A*2079 was found to be able to present A*0201- restricted peptides to and induce the response of CTL, thus it can be classified as member of the HLA-A2 functional supertype family. This finding would benefit the design of peptide vaccines to be applied in broader populations.The nucleotide sequence data reported in this paper have been submitted to the Genbank nucleotide sequence database and have been assigned the accession numbers AY856830 and HWS10002813.The name HLA-A*0279 was officially assigned by the World Health Organization Nomenclature Committee in January 2005.     

Although divergent in residues of the peptide binding site, conserved chimpanzee Patr-AL and polymorphic human HLA-A*02 have overlapping peptide-binding repertoires

Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ～50% of chimpanzee MHC haplotypes. Comparing Patr-AL(+) and Patr-AL(-) haplotypes showed Patr-AL defines a unique 125-kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5' part of human pseudogene HLA-Y, carried by ～10% of HLA haplotypes. Thus, the AL gene alternatively evolved in these closely related species to become classical, nonclassical, and nonfunctional. Although differing by 30 aa substitutions in the peptide-binding α(1) and α(2) domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable with that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α(2) helix. Stimulating PBMCs from Patr-AL(-) chimpanzees with B cells expressing Patr-AL produced potent alloreactive CD8 T cells with specificity for Patr-AL and no cross-reactivity toward other MHC class I molecules, including HLA-A*02. In contrast, PBMCs from Patr-AL(+) chimpanzees are tolerant of Patr-AL.     

Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells

The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.     

Tapasin is a facilitator, not an editor, of class I MHC peptide binding

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.     

Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a StepRank scheme

Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well.     

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

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, gp100(209-217), 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; gp100(209-2M)). The T2M-modified peptide, which is more immunogenic in vitro and in vivo, binds HLA-A2 with a approximately 9-fold greater affinity and has a approximately 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 gp100(209-2M) 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.     

How a T cell receptor-like antibody recognizes major histocompatibility complex-bound peptide

We determined the crystal structures of the T cell receptor (TCR)-like antibody 25-D1.16 Fab fragment bound to a complex of SIINFEKL peptide from ovalbumin and the H-2K(b) molecule. Remarkably, this antibody directly "reads" the structure of the major histocompatibility complex (MHC)-bound peptide, employing the canonical diagonal binding mode utilized by most TCRs. This is in marked contrast with another TCR-like antibody, Hyb3, bound to melanoma peptide MAGE-A1 in association with HLA-A1 MHC class I. Hyb3 assumes a non-canonical orientation over its cognate peptide-MHC and appears to recognize a conformational epitope in which the MHC contribution is dominant. We conclude that TCR-like antibodies can recognize MHC-bound peptide via two different mechanisms: one is similar to that exploited by the preponderance of TCRs and the other requires a non-canonical antibody orientation over the peptide-MHC complex.     

Improving MHC binding peptide prediction by incorporating binding data of auxiliary MHC molecules

MOTIVATION: Various computational methods have been proposed to tackle the problem of predicting the peptide binding ability for a specific MHC molecule. These methods are based on known binding peptide sequences. However, current available peptide databases do not have very abundant amounts of examples and are highly redundant. Existing studies show that MHC molecules can be classified into supertypes in terms of peptide-binding specificities. Therefore, we first give a method for reducing the redundancy in a given dataset based on information entropy, then present a novel approach for prediction by learning a predictive model from a dataset of binders for not only the molecule of interest but also for other MHC molecules. RESULTS: We experimented on the HLA-A family with the binding nonamers of A1 supertype (HLA-A*0101, A*2601, A*2902, A*3002), A2 supertype (A*0201, A*0202, A*0203, A*0206, A*6802), A3 supertype (A*0301, A*1101, A*3101, A*3301, A*6801) and A24 supertype (A*2301 and A*2402), whose data were collected from six publicly available peptide databases and two private sources. The results show that our approach significantly improves the prediction accuracy of peptides that bind a specific HLA molecule when we combine binding data of HLA molecules in the same supertype. Our approach can thus be used to help find new binders for MHC molecules.