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.

Characterization of a Permissive Epitope Insertion Site in Adenovirus Hexon

“…Those results are confirmed here, since antibodies could be generated against PA30 (amino acids 671 to 700 of PA) when it was inserted into HVR5. However, in studies that defined CD4 ϩ T-cell epitopes of PA, no T-cell epitopes were found encompassing residues 671 to 700 (40). We thus sought to characterize the T-cell response to PA30 in the context of HVR5 to determine if it was able to be processed such that it could activate cellular immunity.…”

Asymptomatic HIV-infected Individuals on Antiretroviral Therapy Exhibit Impaired Lung CD4+T-Cell Responses to Mycobacteria

“…We speculate that impaired AM proteolysis affects antigen processing and presentation (21), resulting in suboptimal initiation of antigen-specific alveolar CD4 1 T-cell responses. Impaired AM innate immune functions may, therefore, leave HIV-infected individual on ART vulnerable to LRTI.…”

Sequential Proteolytic Processing of the Capsular Caf1 Antigen of Yersinia pestis for Major Histocompatibility Complex Class II-restricted Presentation to T Lymphocytes

“…However, few candidate vaccine antigens have been sub-jected to a detailed study of the mechanisms of antigen presentation of multiple epitopes (34,35). We investigated antigen presentation of rCaf1 to helper T cells and observed major differences between epitopes depending on their location within the recently solved Caf1 structure (10).…”