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1.
J Immunol ; 164(12): 6120-9, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10843661

ABSTRACT

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/virology , Epitopes, T-Lymphocyte/immunology , HLA-B27 Antigen/immunology , Herpesvirus 4, Human/immunology , Immunodominant Epitopes/immunology , Alleles , Antigen Presentation , B-Lymphocytes/metabolism , Cell Line, Transformed , Clone Cells , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/isolation & purification , Epitopes, T-Lymphocyte/metabolism , HLA-B27 Antigen/biosynthesis , HLA-B27 Antigen/metabolism , Humans , Immunodominant Epitopes/isolation & purification , Immunodominant Epitopes/metabolism , Lymphocyte Activation , Oligopeptides/immunology , Oligopeptides/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Cytotoxic/virology
2.
J Immunol ; 161(1): 112-21, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9647214

ABSTRACT

Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.


Subject(s)
Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/physiology , Cytosol/enzymology , Cytosol/immunology , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/metabolism , Histocompatibility Antigens Class I/metabolism , Multienzyme Complexes/metabolism , Multienzyme Complexes/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/physiology , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Amino Acid Sequence , Animals , Cell Line , Cell-Free System/immunology , Cysteine Endopeptidases/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Cytotoxicity, Immunologic/drug effects , Epitopes, T-Lymphocyte/biosynthesis , Epitopes, T-Lymphocyte/drug effects , Glycine/pharmacology , HLA-A Antigens/biosynthesis , HLA-A Antigens/drug effects , Humans , Leupeptins/pharmacology , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Multienzyme Complexes/drug effects , Proteasome Endopeptidase Complex , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/immunology , Time Factors , Viral Matrix Proteins/biosynthesis , Viral Matrix Proteins/immunology
3.
Int Immunol ; 7(4): 597-605, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7547687

ABSTRACT

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.


Subject(s)
HLA-A2 Antigen/genetics , Immunodominant Epitopes/immunology , Influenza A virus/immunology , T-Lymphocytes, Cytotoxic/immunology , Viral Nonstructural Proteins/immunology , Amino Acid Sequence , Animals , HLA-A2 Antigen/immunology , Humans , Mice , Mice, Transgenic/immunology , Molecular Sequence Data , Peptides/immunology , Viral Nonstructural Proteins/analysis
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