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V H Engelhard

Publications and source records attributed to V H Engelhard.

At least 55 records · Page 3Linked to original sources
PubMed1997

Differential contact of disparate class I/peptide complexes as the basis for epitope cross-recognition by a single T cell receptor.

In an effort to better understand functional recognition of structurally dissimilar ligands by a single TCR, a model system for studying cross-recognition of disparate peptide/class I complexes was developed using the murine (H-2b) CTL clone AHIII12.2, which is reactive to a human self-peptide (p1049) bound to an HLA-A2.1 molecule. We identified a second complex comprised of a synthetic peptide, designated p1058, bound to H-2Db that is recognized by clone AHIII12.2. In cytolysis assays, dose-response profiles for peptides p1049 and p1058 pulsed onto the appropriate target cells were comparable, suggesting that p1049/A2.1 and p1058/Db form functionally equivalent epitopes. To probe the interaction between each complex and the TCR of AHIII12.2, singly substituted analogues of each peptide were tested for their activity in lysis assays. Differences were observed between the two epitopes with respect to permissible residue substitutions at each peptide position from P3 to P8; marked differences were evident at P3 and at P8. The results obtained suggest that this TCR forms critical contacts with atoms at peptide positions P3 and P5 of p1049/A2.1 and at P5 and P8 of p1058/Db, and that TCR cross-recognition of these ligands is a function of both shared and complex-specific contacts made with each epitope. These findings further highlight the versatile reactivity that may be shown by a single TCR and suggest a basis for the recognition of peptide ligands sharing only a limited set of structural features.

Animals↗
PubMed1997

The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

Amino Acid Sequence↗
PubMed1997

The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition.

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Animals↗
PubMed1996

Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100.

Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.

Amino Acid Sequence↗
PubMed1996

Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen.

Hematopoietic chimerism has been used in the laboratory to induce life-long immunologic tolerance to donor antigens. The present study demonstrates that mice transplanted with autologous bone marrow cells retrovirally transduced to express HLA-A2.1 develop a significantly depressed immune response to this antigen while retaining normal reactivity to HLA-B7. Retrovirus-mediated transduction was performed using whole bone marrow-producer cell coculture. This approach did not result in significant gene transfer into hematopoietic progenitor cells. Despite this, the antibody response to HLA-A2.1 in mice reconstituted with genetically modified BMC was completely suppressed three months following bone marrow transplantation. Cell-mediated immunity to HLA-A2.1 was partially suppressed in three-fourths of animals tested three months later, although one animal had a CTL profile similar to that an of HLA-A2.1 transgenic mouse. Complete suppression of the antibody-mediated immune response occurred when only one-third of mice had evidence of the introduced genes in their spleen and one-tenth had the introduced sequences in their circulating WBCs by PCR. In conclusion, engineering of BMC to express donor MHC genes may be an alternative to xenogeneic BMT to induce chimerism and tolerance. More efficient transduction of bone marrow progenitor cells may result in more persistent gene expression and long-lasting transplantation tolerance in recipients of genetically modified bone marrow. Successful application of this technology may also be useful in altering immune responses to other external and self antigens.

Animals↗
PubMed1996

Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.

Antibodies, Monoclonal↗
PubMed1996

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

Animals↗
PubMed1996

Importance of MHC class 1 alpha2 and alpha3 domains in the recognition of self and non-self MHC molecules.

The importance of the species of different domains of class I MHC molecules in peripheral T cell recognition and positive and negative selection was evaluated in a single system. In transgenic mice expressing AAD (containing the alpha1+alpha2 domains of HLA-A2.1 and the alpha3 domain of H-2Dd), the CTL response to influenza peptide M1(58-66) in the context of the alpha1+alpha2 domains of HLA-A2.1 was as strong as the influenza-specific H-2Db-restricted response. However, this strong response was only discernible if the target cell MHC molecule also contained a murine alpha3 domain. In contrast, the response in HLA-A2.1 transgenic mice was about 30-fold weaker, and these CTL were indifferent to the origin of the target molecule alpha3 domain. Further analysis suggested that the major impact of the murine alpha3 domain of the transgene product was to enhance positive selection of a low affinity population of AAD-restricted T cells, presumably through species-specific interaction with CD8. Surprisingly, the response to non-self human class I MHC determinants was not augmented in AAD mice, indicating that the T cells selected are narrowly focused on AAD-related structures. Further analysis indicated that the alphal+alpha2 domains as well as the alpha3 domain influenced the magnitude of the response to non-self human class I MHC determinants, and this effect was mapped to alpha2. We suggest that the alpha2 domains of murine class I molecules contain conserved structural elements that augment the avidity of T cell-class I interactions, and this is particularly important in the recognition of non-self MHC molecules.

Animals↗
PubMed1996

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1996

Identification of a population of CD4+ CTL that utilizes a perforin- rather than a Fas ligand-dependent cytotoxic mechanism.

Although cytotoxic activity has generally been shown to reside in the CD8+ population of T lymphocytes, there are numerous examples of cytotoxic CD4+ T cells. In the present study, we found that after depletion of CD8+ T cells, CD4+ T cells cultured in short-term in vitro mixed lymphocyte cultures developed strong Ag-specific cytotoxic activity. Such CD4+ CTL lysed both Fas+ and Fas- target cells and developed in Fas ligand-deficient gld mice. Thus, in contrast to several recent reports involving long-term T cell clones or short-term mitogen-activated splenocytes, the cytotoxic activity of these CD4+ CTL is not dependent on Fas-Fas ligand interactions. However, CD4+ cells derived in a similar manner from perforin knockout mice showed greatly decreased target cell lysis, indicating that their cytotoxic activity is primarily dependent on a perforin-based mechanism. The Fas-Fas ligand pathway has been shown to be important in the maintenance of peripheral tolerance, but to have a minimal role in protection against viral and bacterial pathogens. Thus, the existence of a population of CD4+ CTL that utilizes perforin re-emphasizes the likely importance of these cells in a classical defensive role against foreign pathogens, in addition to their already implied role as modulators of the immune response.

Animals↗
PubMed1996

Conservation of minor histocompatibility antigens between human and non-human primates.

It is well accepted that minor histocompatibility antigens (mHag) can function as transplantation barriers between HLA-matched individuals. Little is known about the molecular nature and evolutionary conservation of mHag. It is only very recently that the first human mHag were identified. The HLA-A2.1-restricted mHag HA-2 and the HLA-B7-restricted mHag H-Y appeared to be peptides derived from polymorphic self proteins. Here we show that the HLA-A2.1-restricted mHag HA-1, HA-2, and the H-Y peptides are conserved between man, chimpanzees and rhesus macaques. Human cytotoxic T cell clones specific for the HLA-A2.1-restricted mHag HA-1, HA-2, and H-Y recognized HLA-A2.1 gene-transfected chimpanzee and rhesus macaque cells. High-pressure liquid chromatography fractionation of HLA-A2.1-bound peptides isolated from the HLA-A2.1-transfected chimpanzee cells revealed that the chimpanzee HA-1 and HA-2 co-eluted with the human HA-1 and HA-2. Subsequent amino acid sequencing showed that the chimpanzee HA-2 peptide is identical to the human HA-2 peptide. Our functional and biochemical results demonstrate that mHag peptides are conserved for over 35 million years.

Amino Acid Sequence↗
PubMed1996

A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class Ib molecule.

Polymorphism of MHC class Ia molecules severely constrains vaccine development against intracellular pathogens. Antigen presentation by MHC class Ib molecules, which are generally conserved between different individuals, may circumvent this obstacle. Herein, we use tandem mass spectrometry to identify a Listeria monocytogenes pentapeptide antigen that is presented to T lymphocytes by the H2-M3 MHC class Ib molecule. The peptide contains N-formyl methionine at the N terminus and exclusively hydrophobic amino acids. Mice of the H-2 d, H-2 b,and H-2 k haplotypes respond to this peptide upon infection with Listeria monocytogenes. Identification of antigens presented by MHC class Ib molecules is feasible and may provide opportunities for relatively unrestricted vaccine development.

Alleles↗
PubMed1995

Identification of a graft versus host disease-associated human minor histocompatibility antigen.

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.

Amino Acid Sequence↗
PubMed1995

CTL responses of HLA-A2.1-transgenic mice specific for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1.

Vaccine development in animal models depends on ability to recognize epitopes seen by human T cells. In this work, we show that CTL responses in transgenic mice expressing human HLA-A2.1 prospectively predict the same four of 11 hepatitis C virus (HCV) structural protein-derived peptides, expressing a sequence motif for HLA-A2.1 binding, that are actually recognized by human A2.1-restricted CTLs. The CTLs also recognized targets endogenously expressing these proteins. Human CTLs from HCV-infected patients, tested by using the same peptides, revealed a virtually identical response repertoire. A highly conserved HCV core peptide was the most immunogenic, and may be a valuable component of a vaccine against a broad range of HCV isolates in HLA-A2-positive patients. These results suggest that, in spite of species differences, the T cell repertoire is plastic enough to allow a similar response when the same class I MHC molecule is presenting the peptide. Thus, the HLA molecule plays the primary role in determining which peptides are recognized by CTLs. This transgenic mouse model is important for the study of HLA-restricted CTL determinants and for an approach to design a potential HCV vaccine.

Adult↗
PubMed1995

Several HLA alleles share overlapping peptide specificities.

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.

Alleles↗
PubMed1995

Definition of a human T cell epitope from influenza A non-structural protein 1 using HLA-A2.1 transgenic mice.

Previous results from this laboratory demonstrated that the dominant influenza A epitope recognized by HLA-A2.1-restricted cytotoxic T lymphocytes (CTL) from HLA-A2.1 transgenic mice was the matrix protein 1 (M1) peptide epitope that is immunodominant in human CTL responses. However, analysis of a large number of CTL lines revealed a subset of influenza A/PR/8/34-specific murine CTL that recognized an HLA-A2.1-restricted epitope distinct from M1. Using recombinant vaccinia viruses encoding different influenza gene segments, the epitope recognized by these CTL was shown to be derived from A/PR/8 non-structural protein 1 (NS1). Because these CTL did not recognize targets infected with the A/Alaska/6/77 strain of influenza, candidate peptide epitopes were synthesized based on sequences that included an HLA-A2.1-specific binding motif, and that differed between A/PR/8 and A/Alaska. All of these CTL recognized a nonamer and a decamer peptide which contained a common eight amino acid sequence and two distinct sets of binding motif residues. However, the nonamer peptide was able to sensitize CTL for half-maximal lysis at 80- to 2500-fold lower doses than either the octamer or decamer. The homologous peptide derived from A/Alaska NS1 contained conservative amino acid changes at positions 4 and 8, and was not recognized at any tested concentration, although it bound with higher affinity to HLA-A2.1 than the peptide from A/PR/8. The A/PR/8 NS1 nonamer epitope was also recognized by human influenza A-specific CTL derived from two individuals. These results substantiate the general utility of HLA class I transgenic mice for the identification of human CTL epitopes for other pathogens.

Amino Acid Sequence↗
PubMed1994

Diversity and dominance among TCR recognizing HLA-A2.1+ influenza matrix peptide in human MHC class I transgenic mice.

The TCR structures of CTL derived from HLA-A2.1 transgenic mice were analyzed to determine features important in the interaction of murine TCR with the HLA-A2.1 + influenza M1(57-68) peptide complex. V beta 8.1 was dominant in 9 of 11 murine CTL lines, although three other V beta segments were also represented. Sequencing of TCR cDNA from a group of six independently derived CTLs that were V beta 8.1-positive demonstrated a restricted set of D-N-J beta sequences and an apparently restricted set of alpha-chains. However, at least five other distinct alpha beta pairs were found among HLA-A2.1 + M1 peptide-specific CTL in the absence of these chains. Consideration of all TCR sequences obtained demonstrated diverse beta-chain CDR3 regions with some restriction in V alpha segment usage and bias in amino acid sequence of alpha-chain CDR3 regions. Nevertheless, the strongest correlation with HLA-A2.1 + M1 specificity was clearly V beta 8.1 usage. Comparison with previously identified human TCR sequences specific for the same Ag-MHC complex revealed that the dominant murine V alpha and V beta segments used were not the homologues of the dominant human V beta and V alpha segments used. These results together with the lack of interspecies conservation in the alpha- and beta-chain CDR3 regions demonstrate that the dominant TCR structures recognizing HLA-A2.1 + M1(57-68) are substantially different between mouse and humans. Different factors may influence Ag-driven selection of the dominant TCRs used in each species.

Amino Acid Sequence↗