Identification of a novel immunogenic HLA-A*0201-binding epitope of HER-2/neu with potent antitumor properties

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for MHC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/neu decamer LIAHNQVRQV spanning residues 85-94 (HER-2(10(85))). HER-2(10(85)) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(10(85)) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(10(85))-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the rapid expansion of HLA-A2.1/HER-2(10(85))pentamer+/CD8+ cells (PENT+/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(10(85))-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-gamma intracellular staining and in the high sensitivity CD107alpha degranulation assay. Finally, HER-2(10(85)) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(10(85)) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(10(85)) as a possible target for anticancer immunotherapy.     

Class I major histocompatibility complex anchor substitutions alter the conformation of T cell receptor contacts

An immunogenic peptide (GP2) derived from HER-2/neu binds to HLA-A2.1 very poorly. Some altered-peptide ligands (APL) of GP2 have increased binding affinity and generate improved cytotoxic T lymphocyte recognition of GP2-presenting tumor cells, but most do not. Increases in binding affinity of single-substitution APL are not additive in double-substitution APL. A common first assumption about peptide binding to class I major histocompatibility complex is that each residue binds independently. In addition, immunologists interested in immunotherapy frequently assume that anchor substitutions do not affect T cell receptor contact residues. However, the crystal structures of two GP2 APL show that the central residues change position depending on the identity of the anchor residue(s). Thus, it is clear that subtle changes in the identity of anchor residues may have significant effects on the positions of the T cell receptor contact residues.     

T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment

A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.     

Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.     

T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.     

Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/neu-transgenic mice

The HER-2/neu (neu-N)-transgenic mice are a clinically relevant model of breast cancer. They are derived from the parental FVB/N mouse strain and are transgenic for the rat form of the proto-oncogene HER-2/neu (neu). In this study, we report the identification of a MHC class I peptide in the neu protein that is recognized by CD8(+) T cells derived from vaccinated FVB/N mice. This 10-mer was recognized by all tumor-specific FVB/N T cells generated regardless of the TCR Vbeta region expressed by the T cell or the method of vaccination used, establishing it as the immunodominant MHC class I epitope in neu. T cells specific for this epitope were able to cure FVB/N mice of transplanted neu-expressing tumor cells, demonstrating that this is a naturally processed peptide. Altered peptide analogs of the epitope were analyzed for immunogenicity. Vaccination with dendritic cells pulsed with a heteroclitic peptide provided FVB/N and neu-N mice with increased protection against tumor challenge as compared with mice immunized with dendritic cells loaded with either wild-type or irrelevant peptide. Discovery of this epitope allows for better characterization of the CD8(+) T cell responses in the neu-N mouse model in which neu-specific tolerance must be overcome to produce effective antitumor immunity.     

Specificity of peptide binding by the HLA-A2.1 molecule

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.     

Modification of a tumor-derived peptide at an HLA-A2 anchor residue can alter the conformation of the MHC-peptide complex: probing with TCR-like recombinant antibodies

A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC-restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity.     

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

C-terminal anchoring of a peptide to class II MHC via the P10 residue is compatible with a peptide bulge.

The binding of antigenic peptide to class II MHC is mediated by hydrogen bonds between the MHC and the peptide, by salt bridges, and by hydrophobic interactions. The latter are confined to a number of deeper pockets within the peptide binding groove, and peptide side chains that interact with these pockets are referred to as anchor residues. T cell recognition involves solvent-accessible peptide residues along with minor changes in MHC helical pitch induced by the anchor residues. In class I MHC there is an added level of epitope complexity that results from binding of longer peptides that bulge out into the solvent-accessible, T cell contact area. Unlike class I MHC, class II MHC does not bind peptides of discrete length, and the possibility of peptide bulging has not been clearly addressed. A peptide derived from position 24-37 of integrin beta(3) can either bind or not bind to the class II MHC molecule HLA DRB3*0101 based on a polymorphism at the P9 anchor. We show that the loss of binding can be compensated by changes at the P10 position. We propose that this could be an example of a class II peptide bulge. Although not as efficient as P9 anchoring, the use of P10 as an anchor adds another possible mechanism by which T cell epitopes can be generated in the class II presentation system.     

HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy

Tolerance to tumor-nonmutated self proteins represents a major obstacle for successful cancer immunotherapy. Since this tolerance primarily concerns dominant epitopes, we hypothesized that targeting cryptic epitopes that have a low affinity for HLA could be an efficient strategy to breach the tolerance to tumor Ags. Using the P1Y heteroclitic peptide approach, we identified low affinity cryptic HLA-A*0201-restricted epitopes derived from two widely expressed tumor Ags, HER-2/neu and hTERT. The P1Y variants of four HER-2/neu (neu(391), neu(402), neu(466), neu(650))- and two hTERT (hTERT(572) and hTERT(988))-derived low affinity peptides exhibited strong affinity for HLA-A*0201 and stimulated specific CTL from healthy donor PBMCs. These CTL specifically recognized HER-2/neu- and hTERT-expressing tumor cells of various histological origins. In vivo studies showed that HLA-A*0201 transgenic HHD mice vaccinated with the P1Y variant peptides generated CTL that specifically lysed Ag-expressing tumor cells, thus recognizing the cognate endogenous Ags. These results suggest that heteroclitic variants of low affinity, cryptic epitopes of widely expressed tumor Ags may serve as valid tools for tumor immunotherapy.     

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.     

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

Structural and immunological reactivity of the principal neutralizing determinant V3 of glycoprotein gp 120 of HIV-1

The variable domain V3 in the outer glycoprotein gp 120 of HIV-1 is a highly important region with respect to immune response during the course of viral infection. Neutralizing antibodies are produced against this domain; in addition, it has been shown to be a functionally active epitope for T helper and cytotoxic T cells. The high degree of amino acid variability in individual HIV-isolates, however, limits the use of the V3-domain in approaches to vaccine development. In order to characterize the residues important for antibody interaction and binding to MHC class I proteins, we constructed a consensus sequence of the V3-domain with broad reactivity [1] and used synthetic peptides derived from this consensus with individual residues altered to alanine. These peptides were used as antigens in ELISA tests to define the amino acids which are important for binding to human and rabbit/anti-peptide immunoglobulins. In addition, we used these alanine-derived peptides in interaction studies with human HLA-A2.1 and mouse H-2D^d by testing their capacity to stabilize the respective MHC class I protein complexes on the surface of mutant cell lines T2 and RMA-S transfected with D^d gene. The experimental tests allowed us to define individual residues involved in antibody and MHC-protein interaction, respectively. In a further approach, we used those results to design interaction models with HLA-A2.1 and H-2D^d. Therefore, a structural model for H-2D^d was built that exhibits an overall similar conformation to the parental crystal structure of HLA-A2.1. The resulting interaction models show V3-peptide bound in an extended β-conformation with a bulge in its centre for both H-2D^d and HLA-A2.1 complexes. The N- and C-termini of V3 peptide reside in conserved pockets within both MHC-proteins. Anchoring residues could be determined that are crucial for the binding of the respective MHC class I haplotype. The cross-reactivity of V3-peptide in enhancing the expression of two different MHC class I molecules (H-2D^d and HLA-A2.1) is shown to be based on similar peptide binding that induces an almost identical peptide conformation.     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis>(776-90) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/<Emphasis Type="Italic">neu</Emphasis>+ tumors

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.     

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.     

Disulfide bond engineering to trap peptides in the MHC class I binding groove

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells-particularly when the antigenic peptide has relatively weak affinity for the MHC.     

Improved immunogenicity of an immunodominant epitope of the HER-2/neu protooncogene by alterations of MHC contact residues

The HER-2/neu (HER-2) oncogene is expressed in normal epithelial surfaces at low levels and overexpressed in several types of tumors. The low immunogenicity against this self tumor Ag can be improved by developing epitopes with amino acid replacements in their sequences. In this study, three HER-2/neu.369 (HER-2.369) analogue peptides, produced by modifying both anchor positions by introducing L, V, or T at position 2 and V at the C terminus, were analyzed for their capacity to induce CTLs in vitro from human PBMC and in vivo in HLA-A2.1/Kb transgenic mice. One of the analogues (HER-2.369 V2V9) sensitized target cells for HER-2-specific recognition by human CTLs and induced specific CTLs in vitro at 100-fold lower concentrations than the HER-2.369 wild-type epitope. These CTLs were also able to recognize the wild-type epitope and HER-2-expressing tumors in an MHC-restricted manner. Furthermore, a 100-fold lower amount of the HER-2.369 V2V9 analogue compared with the wild-type epitope was required to induce CTLs in HLA-A2.1/Kb transgenic mice. However, the V2V9 analogue demonstrated only marginally better binding to the MHC class I A2 allele compared with wild type. To establish thermodynamic parameters, we developed radiolabeled F3*Y analogues from both the HER-2.369 epitope and the V2V9 analogue. Our results indicate that the high biological activity of the HER-2.369 V2V9 epitope is associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2 stability.     

Increased CD8+ cytotoxic T cell responses to myelin basic protein in multiple sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8(+) T cell responses and their functional properties in patients with MS. There were significantly increased CD8(+) T cell responses to 9-mer MBP peptides, in particular MBP(111-119) and MBP(87-95) peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8(+) T cell lines were of the Th1 phenotype, producing TNF-alpha and IFN-gamma and belonged to a CD45RA(-)/CD45RO(+) memory T cell subset. Further characterization indicated that the CD8(+) T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP(111-119)) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8(+) T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8(+) CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.     

Dissection of the interaction of the human cytomegalovirus-derived US2 protein with major histocompatibility complex class I molecules: prominent role of a single arginine residue in human leukocyte antigen-A2

Human cytomegalovirus encodes several proteins that interfere with expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The unique short protein 2 (US2) binds to many MHC class I allomorphs in the endoplasmic reticulum, preventing cell surface expression of the class I molecule in question. The molecular interactions underlying US2 binding to MHC class I molecules and its allele specificity have not been fully clarified. In the present study, we first compared the sequences and the structures of US2 retained versus non-retained human leukocyte antigen (HLA) class I allomorphs to identify MHC residues of potential importance for US2 binding. On the basis of this analysis, 18 individual HLA-A2 mutants were generated and the ability of full-length US2 to bind wild-type and mutated HLA-A2 complexes was assessed. We demonstrate that Arg181 plays a critical role in US2-mediated inhibition of HLA-A2 cell surface expression. The structural comparison of all known crystal structures of HLA-A2 either alone, or in complex with T cell receptor or the CD8 co-receptor, indicates that binding of US2 to HLA-A2 results in a unique, large conformational change of the side chain of Arg181. However, although the presence of Arg181 seems to be a prerequisite for US2 binding to HLA-A2, it is not sufficient for binding to all MHC class I alleles.