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H M Grey

Publications and source records attributed to H M Grey.

At least 37 records · Page 2Linked to original sources
PubMed1996

Antigen analogs as tools to study T-cell activation function and activation.

Herein we review data relating to the molecular mechanism of T-cell receptor antagonists, their effect on thymic education, the occurrence of naturally occurring antigen analogs, and the therapeutic potential of the TCR antagonist approach. Our data suggest that MHC bound antigen analogs which antagonize the mature T-cell response, bind the T-cell receptor below a crucial affinity threshold required to stimulate the early biochemical events necessary for activation, such as phosphatidylinositol metabolism and the Ca2+ influx. The same peptides do not inhibit the formation of APC/T-cell complexes, suggesting interference with more complex equilibria such as receptor oligomerization and co-receptor interaction. Our laboratory and others have also begun to investigate how these antigen analogs may affect the processes involved in thymic education. Non-stimulatory antagonist peptides can elicit deletion of CD4+/CD8+ thymocytes suggesting a lower affinity requirement for negative selection than for activation. These data also demonstrate that these antigen analogs can, indeed, act upon immature T cells. We have also reviewed evidence that antagonists may, in fact, occur in nature. Recent data suggest that viral mutations resulting in altered immunodominant epitopes may produce antagonist determinants potentially involved in immunosuppression and viral persistence. Finally, we discuss the therapeutic potential of antigen analogs and TCR antagonism in autoimmune disease and allergy.

Animals↗
PubMed1996

Definition of an HLA-A3-like supermotif demonstrates the overlapping peptide-binding repertoires of common HLA molecules.

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.

Alleles↗
PubMed1996

Identification of A2-restricted hepatitis C virus-specific cytotoxic T lymphocyte epitopes from conserved regions of the viral genome.

We have focused on conserved regions of the hepatitis C Virus (HCV) genome to identify viral peptides that contain HLA class I binding motifs and bind with high affinity to the corresponding purified HLA molecules. Accordingly, we have identified 31 candidate epitopes in the HCV that have the potential to be recognized by either HLA-A1, A2.1-, A3, A11- or A24-restricted cytotoxic T lymphocytes (CTL). Twelve conserved peptides that bind HLA-A2.1 with high or intermediate affinity were tested for immunogenicity in vitro in human primary CTL cultures and in vivo by direct immunization of HLA-A2.1/Kb transgenic mice. Six HLA-A2.1-restricted CTL epitopes were immunogenic in both systems. At least three of these peptide epitopes were endogenously processed and presented for CTL recognition. Overall, these data illustrate the value of this approach for the development of virus-specific, peptide-based vaccines.

Amino Acid Sequence↗
PubMed1995

Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules.

The binding capacity of large sets of peptides corresponding to naturally occurring sequences and carrying previously defined A24-specific motifs was analyzed. It was found that only a minority (9-25%) of the motif-carrying peptides bound the relevant HLA-A molecule with good affinity (IC 50% < or = 50 nM), while the majority of them bound only weakly or not at all (IC 50% > or = 500 nM). By correlating the presence of specific residue types at each position along the peptide sequence with average binding affinity, the prominent influence of specific secondary interactions (secondary anchor residues) was revealed. Moreover, secondary interactions appeared to be size-dependent in that the specific effects detected differed in 9-mer and 10-mer peptide sets. Based on these observations, A24-specific refined motifs were also established for both 9-mer and 10-mer ligands, and their merit was verified by testing the binding capacity of independent sets of synthetic peptides. Such refined motifs should facilitate accurate prediction of potential A24-restricted peptide epitopes. It was also noted that certain crucial secondary interactions appear to be remarkably similar in the case of A24 and other HLA-A molecules previously analyzed (A*0201, A3, A11, and others). This may reflect contributions to binding affinity of relatively invariant residues located within the polymorphic pockets of the HLA binding groove.

Alleles↗
PubMed1995

Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

Amino Acid Sequence↗
PubMed1995

Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype.

Direct MHC binding assays with radiolabeled peptides and HLA class I-expressing mammalian cells such as EBV-transformed B cell lines and PHA-activated blasts have been developed. Significant binding of the radiolabeled probe could be obtained if the target cells were preincubated overnight at 26 degrees C in the presence of beta 2-microglobulin. Under these conditions, up to a few percent of the HLA molecules expressed by either cell type could be bound by the labeled peptides. With these assays, the degree of cross-reactivity of the A*0201-restricted hepatitis B virus core 18-27 peptide with other A2 subtypes was examined. It was determined that this peptide epitope also binds the A*0202, A*0205, and A*0206 but not A*0207 subtypes. Inhibition experiments with panels of synthetic peptide analogues underlined the similar ligand specificities of the HLA-A*0201, A*0202, and A*0205 alleles. Analysis of the polymorphic residues that help form the B and F pockets of various HLA alleles allowed prediction of binding of the hepatitis B virus core 18-27 epitope to two other HLA alleles (HLA-A*6802 and A*6901). Thus, it appears that a family of at least six different HLA-A molecules may share overlapping ligand specificities (aliphatic residues in position 2 and at the C termini). These results suggest that broadly cross-reactive peptide epitopes can be identified and greatly enhance the prospective feasibility of peptide-based vaccination approaches.

Amino Acid Sequence↗
PubMed1995

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides.

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Antigen-Presenting Cells↗
PubMed1995

Epitope selection and development of peptide based vaccines to treat cancer.

Cytotoxic T lymphocytes recognize peptides that associate with class I major histocompatibility complex molecules. Since cytotoxic T cells have the capacity to recognize and destroy tumor cells, identification of epitopes recognized by these cells in tumor-associated antigens would allow the production of compounds for the treatment of cancer. Here we review some of the approaches being explored to identify tumor-associated antigens and to develop peptide-based vaccines that induce cytotoxic T lymphocytes against specific tumors.

Amino Acid Sequence↗
PubMed1995

Interaction of antigenic peptides with MHC and TCR molecules.

Recent studies have demonstrated that antigen analogs can act as powerful and specific inhibitors of T cell activation. Analysis of various experimental systems suggests that this effect might be related to engagement of the T cell receptor (TCR) by low-affinity ligands and that the observed inhibition may be mediated by interference with membrane events crucial for signal transduction, such as proper TCR oligomerization. In addition, experimental data obtained by our laboratory and others suggest that TCR antagonism may be of significant value as a therapeutic approach and as a tool to dissect the process of thymic education.

Animals↗
PubMed1995

Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans.

Our goal is to use peptide epitopes that are recognized by cytotoxic T lymphocytes (CTL) as immunogens for the development of prophylactic and therapeutic vaccines with chronic hepatitis B virus (HBV) infection being our first therapeutic target. Because most CTL peptide epitopes are poor immunogens, we specifically modified them by covalently attaching two additional components: a T helper peptide epitope and two lipid molecules. Using the murine influenza virus CTL epitope NP 147-155 as a model system, we found this construct to be highly immunogenic, and a single injection resulted in memory CTL induction that persisted for > 1 yr. Based on the animal studies, a vaccine was designed and tested for both safety and its ability to induce a primary CTL response in normal subjects. The three vaccine components included HBV core antigen peptide 18-27 as the CTL epitope, tetanus toxoid peptide 830-843 as the T helper peptide, and two palmitic acid molecules as the lipids. A dose escalation trial (5, 50, and 500 micrograms) carried out in 26 normal subjects showed that the vaccine was safe and able to induce a primary HBV-specific CTL response. A dose-response curve was observed and five out of five subjects responded to the 500-micrograms dose.

Adolescent↗
PubMed1995

Class I MHC-peptide interactions: structural requirements and functional implications.

In this chapter, we have defined the structural motifs that dictate the capacity of peptides to bind to five different HLA-A alleles that represent some of the most common alleles found in different ethnic populations. In general, these peptide motifs were very specific for the individual HLA-A alleles, with the exception of HLA-A degree 0301 and HLA-A degree 1101, for which the motifs were very similar. When these motifs were tested against an unbiased and complete set of nonamer peptides derived from human papillomavirus E6 and E7 proteins, it was found that the vast majority of high and intermediate binding peptides contained the appropriate motif. Furthermore, using the dominant anchor residues, together with the amino acid positions that interact with secondary anchor residues, it was possible to predict high and intermediate binders. Finally, the finding that there is a direct correlation between binding affinity for MHC and immunogenicity suggests a practical application of being able to predict those peptides that have a high affinity binding for a particular MHC allele--that is, in the design of peptide based vaccines for prophylactic or therapeutic use.

Alleles↗
PubMed1994

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

Amino Acid Sequence↗
PubMed1994

Failure to demonstrate long-lived MHC saturation both in vitro and in vivo. Implications for therapeutic potential of MHC-blocking peptides.

Peptides that bind with high affinity to class II MHC molecules can inhibit T cell activation both in vitro and in vivo. Thus, they have been suggested as potential therapeutic agents for MHC-associated autoimmune diseases. We have constructed nonnatural peptides with high affinity for certain disease-associated MHC alleles. More specifically, a particular peptide, designated as CY-760.50, was found to have a high binding affinity for DR1, slow dissociation kinetics after binding to MHC, and prolonged stability in human serum. However, when the ability of this peptide to block peptide presentation to an influenza hemagglutinin 307-319 peptide-specific, DR1-restricted T cell clone was examined, it was found that MHC blockade could only be achieved when high concentrations of peptide were present along with Ag in the fluid phase. Thus, pretreatment of APC with MHC class II blocker, followed by removal of unbound blocker, did not result in saturation of MHC molecules, because practically immediate reacquisition of Ag-presenting capacity was observed after removal of fluid phase blocker. The pharmacokinetic behavior and the duration of blocking activity of CY-760.50 were also examined in vivo, taking advantage of the fact that the mouse MHC class II molecule I-Ab also bound CY-760.50 with high affinity. CY-760.50 administered i.v. to C57BL/6 mice was rapidly cleared from the circulation and virtually undetectable in the serum 10 min after injection. This fast clearance rate was paralleled by a similarly short duration of the MHC blockade effect. These in vivo results have implications concerning the biology of peptide-MHC interactions, and suggest that MHC blockade may not be feasible as a therapeutic approach unless effective concentrations of inhibitor can be maintained over extended periods of time in the extracellular fluids.

Amino Acid Sequence↗
PubMed1994

Negative selection of CD4+ CD8+ thymocytes by T-cell receptor peptide antagonists.

Antigen-induced activation of T cells can be specifically inhibited by antigen analogs that have been termed T-cell receptor peptide antagonists. These antagonists appear to act by inducing the formation of nonstimulatory or partially stimulatory complexes between T-cell receptors and the major histocompatibility complex molecules presenting the peptides. Herein, we have investigated the effect of T-cell receptor peptide antagonists on thymocyte negative selection. First, peptide antagonists were identified for the cytochrome c-specific T-cell clone AD10. These peptides were then tested for their ability to induce negative selection in an in vitro model system using thymocytes from mice transgenic for the AD10 T-cell receptor. Though unable to induce mature T-cell activation, the T-cell receptor peptide antagonists induced deletion of CD4+ CD8+ thymocytes. These results suggest that negative selection of CD4+ CD8+ thymocytes can be induced by T-cell receptor interactions of a lower affinity than those required for mature T-cell activation.

Animals↗