title: Differential processing of CD4 T-cell epitopes from the protective antigen of Bacillus anthracis.
abstract: We have mapped CD4+ T-cell epitopes located in three domains of the recombinant protective antigen of Bacillus anthracis. Mouse T-cell hybridomas specific for these epitopes were generated to study the mechanisms of proteolytic processing of recombinant protective antigen for antigen presentation by bone marrow-derived macrophages. Overall, epitopes differed considerably in their processing requirements. In particular, the kinetics of presentation, ranging from 15 (fast) to 120 min (slow), suggested sequential liberation of epitopes during proteolytic processing of the intact PA molecule. Pretreatment of macrophages with ammonium chloride or inhibitors of the major enzyme families showed that T-cell responses to an epitope presented with fast kinetics were unaffected by raising endosomal pH or inhibiting cysteine or aspartic proteinases, suggesting presentation independent of lysosomal processing. In contrast, responses to epitopes presented with slower kinetics were dependent on low pH and the activity of cysteine or aspartic proteinases indicating a requirement for lysosomal processing. In addition, responses to all epitopes, whether their presentation was dependent on low pH or not, were prevented by treatment of macrophages with broad spectrum serine proteinase inhibitors. Thus, our data are consistent with a model of sequential antigen processing within the endosomal system, beginning with a pre-processing step mediated by serine or metalloproteinases prior to further processing by lysosomal enzymes. Rapidly presented epitopes seemed to require only limited proteolysis at earlier stages of endocytosis, whereas the majority of epitopes required more extensive processing by neutral proteinases followed by lysosomal enzymes.
authors.affiliation: Musculoskeletal Research Group, Clinical Medical Sciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Musson
authors.forename: Nicola
authors.initials: N
authors.lastname: Walker
authors.forename: Helen
authors.initials: H
authors.lastname: Flick-Smith
authors.forename: E Diane
authors.initials: ED
authors.lastname: Williamson
authors.forename: John H
authors.initials: JH
authors.lastname: Robinson
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D017951:Antigen Presentation; D000942:Antigens, Bacterial; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D001408:Bacillus anthracis / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry; D001854:Bone Marrow Cells / Q000378:metabolism; D015704:CD4 Antigens / Q000096:biosynthesis* / Q000737:chemistry; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D010618:Phenanthrolines / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D015704:CD4 Antigens; D003470:Culture Media; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D010618:Phenanthrolines; D011994:Recombinant Proteins; C030325:anthrax toxin; D000643:Ammonium Chloride; D045726:Metalloproteases; D016282:Aspartic Acid Endopeptidases; D003545:Cysteine