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.
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.
Many human leukemias are characterized by chromosomal translocations yielding hybrid RNAs capable of encoding fusion chimeric proteins. The unique amino acid sequences found in these oncogenic fusion ...proteins represent true tumor-specific antigens that are potentially immunogenic. Although these leukemia-specific fusion proteins have an intracellular location, they might be recognized immunologically by T lymphocytes if peptides derived from the unique sequences are capable of presentation by the major histocompatibility complex (MHC) molecules on leukemic cells. The ability of a series of synthetic peptides corresponding to the junctional sequences of chronic myelogenous leukemia (CMD-derived bcr-abl and acute promyelocytic leukemia (APL)-derived PML-RARα fusion proteins to bind to purified class I molecules was studied. A series of 152 peptides 8, 9, 10, and 11 amino acids in length, spanning the b3a2 and b2a2 breakpoints for CML and PML-RARα A and B breakpoints for APL were analyzed for HLA A1, A2.1, A3.2, A11, A24, B7, B8, and B27 binding motifs. Twenty-one CML peptides and 4 APL peptides were predicted to be potential HLA class I binders. The peptides were tested for binding to appropriate purified HLA molecules in a competition radioimmunoassay. Four peptides derived from b3a2 CML breakpoint bound with high (<50 nmol/L) or intermediate (≤500 nmol/L) affinity to HLA A3, A11, and B8. None of the CML b2a2 or PML-RARα A or B junctional peptides showed affinity of this magnitude for the HLA class I molecules tested. This is the first evidence that tumor-specific breakpoint peptides can bind human MHC class I molecules and provides a rationale for developing a therapeutic vaccine strategy.
Quantitative assays to measure the binding of defined synthetic antigenic peptides and purified MHC class I molecules are described for several common human HLA-A alleles (A1, A2.1, A3, A11 and A24). ...Under appropriate conditions, the binding of radiolabeled peptides to purified MHC class I molecules is very effective, highly specific, and appears to be dependent on the specific sequence motif of the peptide as defined by critical anchor residue positions. Establishment and optimization of the assay reveals that a relatively high fraction of the MHC class I molecules isolated from EBV transformed B cell line sources is capable of binding exogenously added peptide. Scatchard analysis for all alleles yields 5-10% occupancy values. There is a stringent peptide size requirement that is reflected by the direct influence of peptide length on the binding affinity. The peptide-MHC class I interactions demonstrate remarkable similarity to peptide-MHC class II interactions, both in overall affinity and kinetic behavior. The immunological relevance of the peptide-MHC class I binding assay is also demonstrated by measuring the affinity of a panel of previously described HLA restricted peptides for their HLA restriction element. In 91% (10/11) of the cases, the peptides bound with affinities of 50 nM or less, and in the remaining 9% (1/11) of the cases, in the 50 to 500 nM range. Thus, these data provide the first quantitative estimate of what level of HLA-A binding affinity is associated with a diverse panel of immunodominant CTL epitopes in man.
Recent data demonstrate that HLA class I alleles can be grouped into superfamilies based on similarities of their peptide-binding motifs. In this study, we have tested the immunogenicity and ...antigenicity of peptides capable of degenerate binding to multiple HLA class I molecules of the A3-like superfamily. The assay systems utilized included both primary in vitro cultures of lymphocytes from healthy donors, as well as in vitro restimulation of lymphocytes from HIV-infected individuals. Several of the peptides capable of binding more than one HLA A3-like class I molecule were also found to be immunogenic in the context of this same group of A3-like molecules (degenerate CTL recognition). Furthermore, some of the CTL lines thus generated demonstrated promiscuous recognition of the cognate epitope in the context of MHC molecules from more than one member of the superfamily. The fine Ag specificity of this phenomenon was further analyzed using two promiscuous CTL clones derived from A3 and A11 individuals, respectively, and specific for an epitope in the HIV-1 reverse transcriptase. By the use of single-amino acid-substitution analogues, it was demonstrated that the fine specificity of the TCR is largely maintained between MHC-matched and MHC-mismatched presentation of peptide within the A3-like superfamily. These results indicate that the similar peptide-binding specificities among different members of the A3-like superfamily can be reflected in a remarkable similarity in the peptide-MHC complex structures engaged by the TCR and responsible for T cell activation.
To understand and develop strategies to intervene in autoimmune responses to myelin proteolipid protein (PLP), encephalitogenic epitopes must be identified. To expedite the identification of ...potentially immunogenic and encephalitogenic epitopes of PLP, overlapping synthetic 20-mer PLP peptides covering the whole PLP molecule were screened for their ability to bind to purified mouse I-Ad, I-Ak, and I-As molecules. The peptides that bound to the I-A molecules were tested for their ability to induce immune responses in corresponding inbred mouse strains. Immunogenic peptides were then tested for their ability to induce experimental autoimmune encephalomyelitis. Moderate to strong I-A binding was essential for development of immune responses, but immunogenicity was not sufficient for encephalitogenicity. Rather, encephalitogenic epitopes clustered in three regions of the molecule, namely within residues 40-70, 100-119, and 178-209. These were also the regions of the PLP that showed the greatest promiscuity in binding to I-A molecules. Except for PLP 139-151, which is an encephalitogenic determinant in mice expressing I-As, all encephalitogenic epitopes of PLP previously identified, regardless of their MHC class II restriction, are located within or adjacent to these epitope clusters. None of the encephalitogenic epitopes occur in regions of the molecule that have a high degree of homology with the neuronal M6a protein, a member of the DM20/PLP superfamily. Atypical clinical and histologic patterns of experimental autoimmune encephalomyelitis were observed in some strains of mice sensitized with certain PLP peptides and may reflect induction of T cells with different disease-inducing potentials.
An HLA-A3-like supertype (minimally comprised of products from the HLA class I alleles A3, A11, A31, A∗3301, and A∗6801) has been defined on the basis of (a) structural similarities in the ...antigen-binding groove, (b) shared main anchor peptide-binding motifs, (c) the identification of peptides cross-reacting with most or all of these molecules, and (d) the definition of an A3-like supermotif that efficiently predicts highly cross-reactive peptides. Detailed secondary anchor maps for A3, A11, A31, A∗3301, and A∗6801 are also described. The biologic relevance of the A3-like supertype is indicated by the fact that high frequencies of the A3-like supertype alleles are conserved in all major ethnic groups. Because A3-like supertype alleles are found in most major HLA evolutionary lineages, possibly a reflection of common ancestry, the A3-like supermotif might in fact represent a primeval human HLA class I peptide-binding specificity. It is also possible that these phenomena might be related to optimal exploitation of the peptide specificity by human TAP molecules. The grouping of HLA alleles into supertypes on the basis of their overlapping peptide-binding repertoires represents an alternative to serologic or phylogenetic classification.
Herein we describe the establishment of assays to measure peptide binding to purified HLA-B*0701, -B*0801, -B*2705, -B*3501-03, -B*5401, -Cw*0401, -Cw*0602, and -Cw*0702 molecules. The binding of ...known peptide epitopes or naturally processed peptides correlates well with HLA restriction or origin, underscoring the immunologic relevance of these assays. Analysis of the sequences of various HLA class I alleles suggested that alleles with peptide motifs characterized by proline in position 2 and aromatic or hydrophobic residues at their C-terminus shared key consensus residues at positions 9, 63, 66, 67, and 70 (B pocket) and residue 116 (F pocket). Prediction of the peptide-binding specificity of HLA-B*5401, on the basis of this consensus B and F pocket structure, verified this hypothesis and suggested that a relatively large family of HLA-B alleles (which we have defined as the HLA-B7-like supertype) may significantly overlap in peptide binding specificity. Availability of quantitative binding assays allowed verification that, indeed, many (25%) of the peptide ligands carrying proline in position 2 and hydrophobic/aromatic residues at the C-terminus (the B7-like supermotif) were capable of binding at least three of five HLA-B7-like supertype alleles. Identification of epitopes carrying the B7-like supermotif and binding to a family of alleles represented in over 40% of individuals from all major ethnic groups may be of considerable use in the design of peptide vaccines.
(NZB x NZW) F1 (BWF1) mice develop spontaneous T cell autoimmunity to VH region determinants of syngeneic anti-DNA before the onset of clinical disease. In this study, we characterized the ...immunogenicity, MHC binding, and lymphokine secretion patterns induced by T cell determinants from the VH region of one such anti-DNA mAb (A6.1) and examined their role in the regulation of autoimmunity. Determinants were identified by proliferation of syngeneic splenic T cells from young, unprimed BWF1 mice in response to overlapping 12-mer peptides representing the entire VH region sequence. Immunization of young BWF1 mice with any of three determinants (A6H 34-45 p34, A6H 58-69 p58, and A6H 84-95 p84) elicited proliferative responses upon in vitro recall. Upon immunization with the whole A6.1 molecule, however, proliferative responses could be recalled only to the p58 peptide, defining this as immunodominant. The other two peptides (p34 and p84) elicited minimal or no proliferation and could be termed cryptic. Proliferative responses elicited by the cryptic determinants were restricted by a single class II (I-Ed for p34 and I-Au for p84), whereas the immunodominant p58 determinant was restricted by both I-Ed and I-Eu. The cryptic p34 and p84 bound strongly to I-Ed and I-Au, respectively, whereas the immunodominant p58 peptide bound poorly to I-Ed. A6H p84 elicited T cells that secreted lymphokines in a pattern consistent with a Th1-like phenotype, whereas p58 induced a Th2-like cytokine pattern. Immunization with p34 or p84, or adoptive transfer of a p84-reactive T cell line to young BWF1 mice significantly increased IgG anti-DNA levels, accelerated nephritis, and decreased survival. In conclusion, in BWF1 mice, autoreactive T cells recognizing both cryptic and dominant self-determinants on anti-DNA autoantibodies escape deletion or anergy induction. Furthermore, since these cells are spontaneously activated before the onset of clinical disease, they may be involved in the development of the autoimmune process.
We investigated the immune response to proteolipid protein (PLP), the most abundant central nervous system myelin protein in humans. A total of 8207 short-term T cell lines were generated from 49 ...individuals, 39 patients with multiple sclerosis and 10 control subjects. As we have reported previously, the frequency of PLP-reactive T cells did not differ between the two groups. To determine immunodominant PLP epitopes, proliferative responses of 971 PLP-specific lines were tested with 27 overlapping 20-amino acid peptides encompassing the human PLP sequence and the binding affinities of the PLP peptides to DRB5*0101 and DRB1*1501, DR2 MHC class II isotypes associated with multiple sclerosis, were determined. The T cell response after primary PLP stimulation was focused on two immunodominant epitopes comprising residues p30-49 and p180-199. These two fragments were recognized after processing of native protein by APCs and were situated in hydrophilic regions of PLP exhibiting only moderate affinity to DR2 molecules. In contrast, when T cells from DR2+ subjects were stimulated initially by individual synthetic peptides with either high or low affinity to DRB5*0101 and DRB1*1501 isotypes, additional cryptic epitopes were recognized. MHC restriction of lines specific for the cryptic PLP epitopes were related to binding affinity to DR2 isotypes. Our results indicate that protein Ags are recognized in vivo as immunodominant epitopes after Ag processing by APCs and as cryptic epitopes after processing, presumably by extracellular proteolytic enzymes.
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