Biomedical subjects

E Appella

Publications and source records attributed to E Appella.

At least 91 records · Page 5Linked to original sources

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

Conformation and surface expression of free HLA-CW1 heavy chains in the absence of beta 2-microglobulin.

A beta 2-microglobulin (beta 2m)-deficient kidney carcinoma cell line and three monoclonal antibodies to the alpha 1 (L31), alpha 2 (W6/32), and alpha 3 (Q1/28) domain of class I HLA molecules were selected to assess the role of beta 2m in regulating the conformation and surface expression of HLA-C molecules. HLA-A2, -B27, and -CW1 molecules synthesized by beta 2m-deficient cells were compared to heavy chains synthesized in transfectants expressing a large excess of beta 2m. As assessed by differential binding with monoclonal antibodies and partitioning studies in the detergent TX-114, no HLA-A2, -B27, or -CW1 molecules can be expressed, in a correct conformation, by beta 2m-deficient cells. These cells, however, do express low but significant amounts of free HLA-CW1 heavy chains at the cell surface. Transfection with beta 2m causes a coordinate change in the antibody reactivity of the three domains of HLA-CW1 molecules, thereby providing the first experimental demonstration that assembly with beta 2m affects the folding of not only the alpha 1 and alpha 2, but also of the alpha 3 domain. HLA-CW1 heavy chains, when free of beta 2m, are less soluble in the detergent TX-114 than free HLA-B27 heavy chains, and when associated with beta 2m share an alpha 3 domain epitope with free HLA-A2 and -B27 heavy chains. Moreover, their assembly with beta 2m is largely incomplete. Those data additionally demonstrate an impaired ability of HLA-CW1 to properly fold and establish a close similarity of HLA-CW1 to murine Db and Ld molecules. Although the functional role, if any, of free HLA-CW1 heavy chains remains to be determined, the present study demonstrates that the absence of beta 2m does not completely ablate class I expression in neoplastic cells of human origin.

Carcinoma, Renal Cell↗

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

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↗

PubMed1997

Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles.

We recently described human leukocyte antigen (HLA) A2, A3 and B7 supertypes, characterized by largely overlapping peptide-binding specificities and represented in a high percentage of different populations. Here, we identified 17 Plasmodium falciparum peptides capable of binding these supertypes and assessed antigenicity in both vaccinated and naturally exposed populations. Positive cytotoxic T lymphocyte recall and cytokine (interferon-gamma and tumor necrosis factor alpha) responses were detected for all peptides; all were recognized in the context of more than one HLA class I molecule; and at least 12 of the 17 were recognized in the context of all HLA alleles studied. These data validate the concept of HLA supertypes at the biological level, show that highly degenerate peptides are almost always recognized as epitopes, and demonstrate the feasibility of developing a universally effective vaccine by focusing on a limited number of peptide specificities.

Alleles↗

PubMed1997

Effect of phosphorylation on tetramerization of the tumor suppressor protein p53.

Human tumor suppressor protein p53 is a 393-amino acid phosphoprotein that enhances transcription in response to DNA damage from several genes that regulate cell cycle progression. The tetrameric state of p53 is critical to wild-type function; the p53 tetramerization element is located in the C-terminal region of the protein. This region is phosphorylated at several evolutionarily conserved serines, suggesting that phosphorylation may be an important regulator of p53 function. In order to determine the effect of phosphorylation on tetramer formation, we synthesized phosphopeptides corresponding to p53(Ser303-Asp393) with phosphate incorporated at Ser315, Ser378, or Ser392, and at both Ser315 and Ser392. Equilibrium ultracentrifugation analysis showed that phosphorylation at Ser392 increased the association constant for tetramer formation nearly ten-fold. By itself, phosphorylation at Ser315 or Ser378 had little effect on tetramer formation, but Ser315 largely reversed the effect of phosphorylation at Ser392. Analysis by calorimetry suggests that phosphorylation may influence subunit affinity by an enthalpy driven process.

Amino Acid Sequence↗

PubMed1997

Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.

Trichothiodistrophy (TTD), xeroderma pigmentosum (XP), and Cockayne's syndrome (CS) are three distinct human diseases with sensitivity to ultraviolet (UV) radiation affected by mutations in genes involved in nucleotide excision repair (NER). Among the many responses of human cells to UV irradiation, both nuclear accumulation of p53, a tumor suppressor protein, and alterations in cell-cycle checkpoints play crucial roles. The purpose of this study was to define the signals transmitted after UV-C-induced DNA damage, which activates p53 accumulation in TTD/XP-D fibroblasts, and compare this with XP-D cell lines that carry different mutations in the same gene, XPD. Our results showed that p53 was rapidly induced in the nuclei of TTD/XP-D and XP-D fibroblasts in a dose-dependent manner after UV-C irradiation, as seen in XP-A and CS-A fibroblasts, much lower doses being required for the protein accumulation than in normal human fibroblasts, XP variant cells, and XP-C cells. The kinetics of accumulation of p53 and two effector proteins involved in cell-cycle arrest, WAF1 and GADD45, were also directly related to the repair potential of the cells, as in normal human fibroblasts their levels declined after 24 h, the time required for repair of UV-induced lesions, whereas NER-deficient TTD/XP-D cells showed p53, WAF1, and GADD45 accumulation for over 72 h after irradiation. Our results indicate that p53 accumulation followed by transcriptional activation of genes implicated in growth arrest is triggered in TTD/XP-D cells by the persistence of cyclobutane pyrimidine dimers, which are known to block transcription, on the transcribed strands of active genes.

Adolescent↗

PubMed1996

The oligomerization domain of p53: crystal structure of the trigonal form.

The structure of the oligomerization domain of the p53 tumor suppressor protein was determined in the trigonal crystal form, using a refined NMR structure as a model. A synthetic peptide comprising residues 319-360 of human p53 crystallized in the space group P3(1)21. There is one biologically relevant tetrameric domain in the crystallographic asymmetric unit. The structure was refined jointly with NMR data, only the third such case (the previous examples being IL-1beta (Shaanan, B., Gronenborn, A.M., Cohen, G.H., Gilliland, G.L., Veerapandian, B., Davies, D.R. and Clore, G.M. (1992) Science 257, 961-964 [1]) and BPTI (Schiffer, C., Huber, R., Wuthrich, K. and Van Gunsteren, W.F. (1994) J. Mol. Biol. 241, 588-599 [21)), to 2.5 A resolution with an R factor of 0.207. The distribution of tumor-derived mutations in the oligomerization region together with structural and biological data suggest a strategy for the design of antitumor therapeutics.

Biopolymers↗

PubMed1996

Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes.

The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31-restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

Amino Acid Sequence↗

PubMed1996

Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor.

The cytoplasmic tails of both the beta and gamma subunits of the high affinity IgE receptor (FcepsilonRI) contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the beta and gamma subunits of FcepsilonRI were used to investigate which proteins associate with these motifs. Tyrosine-phosphorylated beta and gamma ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-gamma1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine-diphosphorylated gamma ITAM peptide, but much less by the diphosphorylated beta ITAM peptide or by the monophosphorylated peptides. Phospholipase C-gamma1, Shc, and Grb2 were precipitated only by the diphosphorylated beta ITAM peptide. Non-phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-gamma1, Shc, Syk, and Lyn directly bound the tyrosine-phosphorylated ITAM peptides. Although the ITAM sequences of the beta and gamma subunits of FcepsilonRI are similar, once they are tyrosine-phosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the gamma subunit recruits and activates Syk, whereas the beta subunit may be important for the Ras signaling pathway.

Amino Acid Sequence↗

PubMed1996

Invariant chain and DM edit self-peptide presentation by major histocompatibility complex (MHC) class II molecules.

We have studied the consequences of invariant chain (Ii) and DM expression on major histocompatibility complex (MHC) class II function. Ii has a number of discrete functions in the biology of class II, including competitive blocking of peptide binding in the endoplasmic reticulum and enhancing localization in the endocytic compartments. DM is thought to act primarily in endosomes to promote dissociation of the Ii-derived (CLIP) peptide from the class II antigen-binding pocket and subsequent peptide loading. In this study, we have evaluated the functional role of Ii and DM by examining their impact on surface expression of epitopes recognized by a large panel of alloreactive T cells. We find most epitopes studied are influenced by both Ii and DM. Most strikingly, we find that surface expression of a significant fraction of peptide-class II complexes is extinguished, rather than enhanced, by DM expression within the APC. The epitopes antagonized by DM do not appear to be specific for CLIP. Finally, we found that DM was also able to extinguish recognition of a defined peptide derived from the internally synthesized H-2Ld protein. Thus, rather than primarily serving in the removal of CLIP, DM may have a more generalized function of editing the array of peptides that are presented by class II. This editing can be either positive or negative, suggesting that DM plays a specifying role in the display of peptides presented to CD4 T cells.

Animals↗

PubMed1996

The proteolytic environment involved in MHC class II-restricted antigen presentation can be modulated by the p41 form of invariant chain.

During biosynthesis, MHC class II associates with invariant chain which exists in two forms, p31 and p41. Both forms of invariant chain prevent peptide binding to class II, facilitate transport, and enhance class II localization to Ag-processing compartments. In spite of these shared functions, presentation of some Ags can be selectively enhanced by expression of p41. Here we show that p41 can function as a protease inhibitor: 1) the functional and biochemical consequences of p41 expression can be mimicked by inhibiting cysteine proteases in vivo; 2) the amount of intracellular active cysteine proteases is dramatically decreased in p41-positive cells; and 3) a polypeptide corresponding to the p41-unique region is a potent inhibitor of cathepsin L in vitro. These data suggest that p41 can enhance Ag presentation by reducing the proteolytic activity of the Ag-processing compartment, thus protecting a subset of antigenic epitopes from excessive degradation.

Animals↗

PubMed1996

Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Amino Acid Sequence↗

PubMed1996

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

Amino Acid Sequence↗

PubMed1996

Expression of IFN regulatory factor family proteins in lymphocytes. Induction of Stat-1 and IFN consensus sequence binding protein expression by T cell activation.

Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the IFN regulatory factor (IRF) family. Evidence indicates that this family has a function in the immune system. Unlike other members of the family, ICSBP is expressed exclusively in the immune system. In this work, immunoblot analysis was performed to study expression of ICSBP and other members of the family in various murine lymphocytes. The results show that all IRF family members are expressed constitutively in B cells throughout development, and in resting and activated cells. In contrast, ICSBP expression was undetectable in thymocytes and resting T cells, while all other IRF proteins tested (IRF-1, IRF-2, and ISGF3-gamma) were detected in these cells. Induction of ICSBP (and weakly IRF-1, but not other members) was observed upon activation of T cells following anti-CD3 Ab binding or Con A stimulation. Once T cells were activated, ICSBP was expressed stably in both Th1 and Th2 cells. We show that Stat-1, which binds to the IFN-gamma-responsive element of the ICSBP promoter, was induced following anti-CD3 Ab and Con A stimulation. Stat-1 induction was found in T cells of IFN-gamma+/+, but not of IFN-gamma-/- mice, indicating that T cell activation stimulates the Stat pathway of transcription that is mediated through IFN-gamma. IFN-gamma-activated Stat-1 partly accounted for ICSBP induction in activated T cells, as levels of induction were lower in IFN-gamma-/- than in IFN+/+ T cells. Taken together, these results show that activation of ICSBP is coupled with T cell activation that is partly due to IFN-gamma-induced Stat-1.

Amino Acid Sequence↗

PubMed1996

Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy.

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.

Alleles↗

PubMed1996

Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.

Amino Acid Sequence↗

PubMed1996

A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes.

A number of antigens recognized by tumor-reactive T cells have recently been identified. The antigens identified in mouse model systems appear, with one exception, to represent the products of mutated genes. In contrast, most of the antigens recognized by human tumor-reactive T cells reported to date appear to represent the products of non-mutated genes. Here we report the isolation of a cDNA clone encoding beta-catenin, which was shown to be recognized by the tumor-infiltrating lymphocyte (TIL) 1290, a HLA-A24 restricted melanoma-specific CTL line from patient 888. The cDNA clone, which was isolated from the autologous melanoma cDNA library, differed by a single base pair from the published beta-catenin sequence, resulting in a change from a serine to a phenylalanine residue at position 37. Normal tissues from this patient did not express the altered sequence, nor did 12 allogeneic melanomas, indicating that this represented a unique mutation in this patient's melanoma. A peptide corresponding to the sequence between amino acids 29 and 37 of the mutant gene product was identified as the T cell epitope recognized by TIL 1290. The observation that HLA-A24 binding peptides contain an aromatic or hydrophobic residue at position 9 suggested that the change at position 37 may have generated a peptide (SYLDSGIHF) which was capable of binding to HLA-A24, and a competitive binding assay confirmed this hypothesis. The beta-catenin protein has been shown previously to be involved in cell adhesion mediated through the cadherin family of cell surface adhesion molecules. The high frequency of mutations found in members of cellular adhesion complexes in a variety of cancers suggests that these molecules may play a role in development of the malignant phenotype.

Amino Acid Sequence↗