Biomedical subjects

A Sette

Publications and source records attributed to A Sette.

At least 127 records · Page 7Linked to original sources

PubMed1997

Induction of antitumor cytotoxic T lymphocytes with a MAGE-3-encoded synthetic peptide presented by human leukocytes antigen-A24.

For the development of immunotherapy using MAGE peptides, the identification of additional tumor antigens is required. Because HLA-A24 is the most common allele in Japanese and is also frequently present in Caucasians, MAGE-3-encoded synthetic peptides with binding affinity for HLA-A24 were thus tested for the induction of specific CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A24 healthy donors using a simplified method. By using a peptide with a sequence of IMPKAGLLI (amino acid position in MAGE-3 195-203), the CTL responses could thus be induced from unseparated PBMCs by stimulation with freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and by also using interleukin 7 and keyhole limpet hemocyanin for a primary culture. The induced CTLs could lyse HLA-A24 carcinoma cells expressing MAGE-3, as well as the peptide-pulsed target cells, in an HLA class-I restricted manner. The identification of the MAGE-3/HLA-A24 peptide, IMPKAGLLI, may thus potentially offer the opportunities to design peptide-based immunotherapeutic approaches that might prove to be effective in treating patients with MAGE-3-positive malignant tumors.

Alleles↗

PubMed1997

Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells.

Human melanoma-specific HLA-A2 restricted CTLs have recently been shown to recognize antigens expressed by melanoma lines and normal melanocytes, including Melan-A/Mart-1, gp100, gp75, and tyrosinase. Herein, we define HLA-A2-restricted CTL epitopes from a recently cloned melanocortin 1 receptor (MC1R), which belongs to a new subfamily of the G-protein-coupled receptors expressed on melanomas and melanocytes. Thirty-one MC1R-derived peptides were selected on the basis of HLA-A2-specific motifs and tested for their HLA-A2 binding capacity. Of a group of 12 high or intermediate HLA-A2 binding peptides, three nonamers, MC1R244 (TILLGIFFL), MC1R283 (FLALIICNA), and MC1R291 (AIIDPLIYA), were found to induce peptide-specific CTLs from peripheral blood mononuclear cells of healthy HLA-A2+ donors after repeated in vitro stimulation with peptide-pulsed antigen-presenting cells. The CTLs raised against these three HLA-A2+-restricted peptides could recognize naturally processed peptides from HLA-A2+ melanomas and from Cos7 cells cotransfected with MC1R and HLA-A2. CTLs induced by the MC1R291 peptide (but not induced or induced only to a very low extent by the other two MCR1 peptide epitopes) showed cross-reactions with two other members of the melanocortin receptor family, which are more broadly expressed on other tissues. Taken together, our findings have implications in relation both to autoimmunity and immunotherapy of malignant melanomas.

Animals↗

PubMed1997

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens.

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139-151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139-151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178-191) and myelin oligodendrocyte protein (MOG) residues 92-106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139-151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139-151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Animals↗

PubMed1997

Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3-like superfamily: implications for vaccine development.

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.

AIDS Vaccines↗

PubMed1997

Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis.

The present study was designed to determine if highly conserved hepatitis B virus (HBV)-derived peptides that bind multiple HLA class I alleles with high affinity are recognized as cytotoxic T lymphocyte (CTL) epitopes in acutely infected patients. Peripheral blood mononuclear cells from 67 patients with acute hepatitis B, and 12 patients convalescent from acute hepatitis B, were stimulated with three panels of peptides, each of which bind with high affinity to several class I alleles from the HLA-A2-, HLA-A3-, or HLA-B7-supertypes. In these patients, 8 of the 19 peptides tested were found to represent CTL epitopes recognized by two or more alleles in each supertype. Two sets of nested peptides were recognized in the context of alleles with completely unrelated peptide binding specificities. Finally, promiscuous recognition by the same CTL of a given peptide presented by target cells expressing different A2 subtypes was also commonly observed. In conclusion, several HBV-specific CTL epitopes, recognized by acutely infected or convalescent patients in the context of a wide range of HLA alleles have been identified. These results demonstrate the functional relevance of the supertype grouping of HLA class I molecules in a human viral disease setting. Furthermore, they represent a significant advance in the development of a totally synthetic vaccine to terminate chronic HBV infection and support the feasibility of a systematic approach to development of similar vaccines for prevention and treatment of other chronic viral infections.

Acute Disease↗

PubMed1997

The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection.

We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.

Acute Disease↗

PubMed1997

Determinants of T cell reactivity to the Mycobacterium leprae GroES homologue.

The 10-kDa protein Ag of Mycobacterium leprae, a human GroES hsp10 cognate, is a major T cell Ag in human leprosy infection. We investigated the mechanism for T cell responsiveness to this Ag according to the trimolecular interaction between T cell, peptide, and Ag-presenting element. This research was accomplished by mapping T cell epitopes in leprosy patients and correlating these responses with peptide-MHC binding affinities. We found that the majority of tuberculoid leprosy patients responded to peptides corresponding to residues 25-39 and 28-42. Truncation analysis of these peptides mapped the exact epitope to be within the overlapping region comprising residues 28-39. Responsiveness was correlated with the HLA-DRB5*0101 allele, which bound the peptides with moderate affinity. This allele is linked to HLA-DR2, which is associated with the resistant form of leprosy. Therefore, T cell responsiveness in tuberculoid leprosy may be mediated by the ability of HLA-DRB5*0101 to bind and present peptides of the immunodominant 10-kDa Ag.

Alleles↗

PubMed1997

Differential effects of cytolytic T cell subsets on intracellular infection.

In analyzing mechanisms of protection against intracellular infections, a series of human CD1-restricted T cell lines of two distinct phenotypes were derived. Both CD4(-)CD8(-) (double-negative) T cells and CD8(+) T cells efficiently lysed macrophages infected with Mycobacterium tuberculosis. The cytotoxicity of CD4(-)CD8(-) T cells was mediated by Fas-FasL interaction and had no effect on the viability of the mycobacteria. The CD8(+) T cells lysed infected macrophages by a Fas-independent, granule-dependent mechanism that resulted in killing of bacteria. These data indicate that two phenotypically distinct subsets of human cytolytic T lymphocytes use different mechanisms to kill infected cells and contribute in different ways to host defense against intracellular infection.

Antigens, CD1↗

PubMed1997

Two complementary methods for predicting peptides binding major histocompatibility complex molecules.

Peptides that bind to major histocompatibility complex products (MHC) are known to exhibit certain sequence motifs which, though common, are neither necessary nor sufficient for binding: MHCs bind certain peptides that do not have the characteristic motifs and only about 30% of the peptides having the required motif, bind. In order to develop and test more accurate methods we measured the binding affinity of 463 nonamer peptides to HLA-A2.1. We describe two methods for predicting whether a given peptide will bind to an MHC and apply them to these peptides. One method is based on simulating a neural network and another, called the polynomial method, is based on statistical parameter estimation assuming independent binding of the side-chains of residues. We compare these methods with each other and with standard motif-based methods. The two methods are complementary, and both are superior to sequence motifs. The neural net is superior to simple motif searches in eliminating false positives. Its behavior can be coarsely tuned to the strength of binding desired and it is extendable in a straightforward fashion to other alleles. The polynomial method, on the other hand, has high sensitivity and is a superior method for eliminating false negatives. We discuss the validity of the independent binding assumption in such predictions.

Artificial Intelligence↗

PubMed1997

Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses.

Certain changes in TCR contact residues have been shown to have profound effects on the capacity of a peptide Ag to stimulate a T cell response. Although some of these changes apparently lead to a complete loss of the ability to interact with the TCR, others result in partial agonist activity (e.g., cytokine production without proliferation) or antagonist activity (i.e., the capacity to inhibit the engagement to the TCR by Ag). We show MHC class II-restricted antagonist activity was associated with a differential pattern of early tyrosine phosphorylation events that was characterized by a preponderance of phosphorylation of low molecular mass TCRzeta and the failure to phosphorylate Zap-70. These early tyrosine phosphorylation patterns are the same as those previously described for partial agonists. Thus, a partial agonist phenotype such as anergy induction cannot be ascribed in a causal manner to this pattern of tyrosine phosphorylation. We further extend the studies of signal transduction elicited by agonist and antagonist peptides by characterizing differential recruitment of Zap-70 associated with TCRzeta isoforms and differential phosphorylation of p120 proto-oncogene c-Cbl. Another early event following TCR engagement by Ag, down-modulation of the TCR, was studied with antagonist peptides. We show that antagonist peptides do not cause TCR down-modulation. This failure may represent a mechanism by which antagonists inhibit antigen-mediated stimulation of T cells.

Amino Acid Sequence↗

PubMed1997

Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells.

The gp100 melanoma-associated tumor Ag was selected as a model system to study the diversity of human antitumor cytotoxic T cell responses. First, peptides corresponding to dominant gp100 HLA-A2.1-restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol that uses peptide-pulsed dendritic cells as APCs and IL-7 and IL-10 as immune-enhancing cytokines. High CTL activity toward both peptide-pulsed target cells and gp100+ melanoma cells was obtained with four out of five peptides tested. Second, HLA-A2.1-binding peptides from gp100 that do not appear to represent CTL epitopes in melanoma patients were also tested for their capacity to induce CTL using the in vitro priming protocol. Three of six peptides tested induced CTL in lymphocytes from normal volunteers. One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission. Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T cell response, they may be considered as typical "subdominant" epitopes. The results are discussed in the context of the usefulness of this approach to detail the immunologic potential of a given tumor-associated Ag and its relevance for the design of effective immune-based therapies.

Antigens, Neoplasm↗

PubMed1997

Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: implications on immunogenicity and immunodominance.

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA-A*0201/Kb (A2/Kb) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA-A*0201-binding motif, we selected A2-binding peptides, immunized A2/Kb animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide-specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/Kb animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide-specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.

Amino Acid Sequence↗

PubMed1997

A DR17-restricted T cell epitope from a secreted Mycobacterium tuberculosis antigen only binds to DR17 molecules at neutral pH.

The assembly of peptide-major histocompatability class II complexes in vitro is accelerated at low pH, comparable to that found in the intracellular compartments of metabolically active antigen-presenting cells (APC). Mycobacteria such as Mycobacterium tuberculosis reside in phagosomes with only mildly acidic pH. Therefore, we investigated the pH dependency of peptide-HLA-DR binding for several T cell epitopes of mycobacterial proteins, focussing particularly on well-defined, immunodominant HLA-DR17(3)-restricted T cell epitopes: peptide (p) 3-13 from the cytoplasmic 65-kDa heat shock protein of M. tuberculosis/M. leprae, and peptide 56-65 from the secreted 30/31-kDa protein from M. tuberculosis/M. leprae. p3-13 bound to purified, cell-free DR17 under both acidic and neutral conditions. Four other, unrelated DR17-binding peptides showed the same pH-dependent binding characteristics as p3-13. p56-65, however, only bound to purified DR17 at pH 7 but not at all at pH 4.5. These DR17 peptide binding data were confirmed in cell-bound DR17, in T cell stimulation assays in which fixed APC were peptide-pulsed at acidic or neutral pH before addition of peptide-specific DR17-restricted T cells. As far as we are aware, p56-65 is the only human T cell epitope binding to HLA exclusively at neutral pH. The binding characteristics of p56-65 may reflect dominant processing in alternative, less acidic vacuolar compartments specifically related to the generation of epitopes from (secreted) mycobacterial proteins. The observation that p56-65 is an immunodominant epitope for anti-mycobacterial T cells suggests the relevance of such novel processing compartments in T cell-mediated immunity.

Amino Acid Sequence↗

PubMed1997

Molecular features of the hepatitis B virus nucleocapsid T-cell epitope 18-27: interaction with HLA and T-cell receptor.

The strength of the cytotoxic T lymphocyte (CTL) response is believed to influence the final outcome of hepatitis B virus (HBV) infection. Among the different CTL epitopes so far identified, the sequence 18-27 of the HBV nucleocapsid antigen is widely recognized by CTL of HLA-A2-positive patients with acute self-limited HBV infection, and represents the main component of a peptide-based therapeutic vaccine aimed at stimulating the antiviral CTL response in patients with chronic hepatitis B. In the present study, we further analyzed the features of this important HBV region by the following: 1) defining the contribution of individual residues of the epitope to the interaction with the T-cell receptor (TCR) and with the HLA-A0201 molecule; 2) assessing the antigenicity of this viral region in the context of the different HLA-A2 subtypes; and 3) testing whether this sequence can stimulate not only HLA-class I but also HLA class II restricted T-cell responses. A clear hierarchy was observed in the ability of individual residues to act as TCR or HLA binding sites. Furthermore, the sequence HBc18-27 was able to be recognized by specific CTL when presented in the context of different HLA-A2 subtypes. Finally, this HBV region was also found to stimulate HLA class II restricted T-cell responses. These data further increase the potential coverage and efficacy of therapeutic vaccines based on the HBc18-27 sequence.

Adult↗

PubMed1997

Two distinct HLA-A*0101-specific submotifs illustrate alternative peptide binding modes.

Previous studies have defined two different peptide binding motifs specific for HLA-A*0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A*0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A*0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE3 submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A*0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A*0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A*0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A*0101-restricted peptide epitopes.

Binding Sites↗

PubMed1997

The HLA-A0207 peptide binding repertoire is limited to a subset of the A0201 repertoire.

Quantitative A*0207 peptide binding assays have been developed utilizing HLA transfected cells and affinity purified molecules. By using a panel of single substitution analog peptides, it was demonstrated that A*0207 binds peptides with main anchor specificity at position 2 and the C-terminus similar to A*0201. Previous data indicating that A*0207 (but not A*0201) also requires the presence of D or P in position 3 of its peptide ligands was confirmed by the analysis of additional single substituted analogs. Finally, by analyzing the A*0201 and A*0207 binding capacities of panels of unrelated synthetic peptides, it was found that 8/15 (53.3%) A*0201 binders with D or P in position 3 bound A*0207, while only 5/72 (6.9%) A*0201 binders without D or P in position 3 also bound A*0207. Together, these data indicate that although A*0207 may be included amongst A2 supertype alleles, its peptide binding repertoire is largely limited to a subset of that bound by A*0201.

Antigen Presentation↗

PubMed1997

A structure-based algorithm to predict potential binding peptides to MHC molecules with hydrophobic binding pockets.

Binding of peptides to MHC class I molecules is a prerequisite for their recognition by cytotoxic T cells. Consequently, identification of peptides that will bind to a given MHC molecule must constitute a central part of any algorithm for prediction of T-cell antigenic peptides based on the amino acid sequence of the protein. Binding motifs, defined by anchor positions only, have proven to be insufficient to ensure binding, suggesting that other positions along the peptide sequence also affect peptide-MHC interaction. The second phase of prediction schemes therefore take into account the effect of all positions along the peptide sequence, and are based on position-dependent-coefficients that are used in the calculation of a peptide score. These coefficients can be extracted from a large ensemble of binding sequences that were tested experimentally, or derived from structural considerations, as in the algorithm developed by us recently. This algorithm uses the coordinates of solved complexes to evaluate the interactions of peptide amino acids with MHC contact residues, and results in a peptide score that reflects its binding energy. Here we present our analysis for peptide binding to four MHC alleles (HLA-A2, HLA-A68, HLA-B27 and H-2Kb), and compare the predictions of the algorithm to experimental binding data. The algorithm performs successfully in predicting peptide binding to MHC molecules with hydrophobic binding pockets but not when MHC molecules with hydrophilic, charged pockets are considered. For MHC molecules with hydrophobic pockets it is demonstrated how the algorithm succeeds in distinguishing binding from non-binding peptides, and in high ranking of immunogenic peptides within all overlapping same-length peptides spanning their respective protein sequences. The latter property of the algorithm makes it a useful tool in the rational design of peptide vaccines aimed at T-cell immunity.

Algorithms↗

PubMed1997

Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper epitopes (PADRE) for antibody responses in vivo.

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

Adjuvants, Immunologic↗