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J. Biol. Chem., Vol. 278, Issue 52, 52425-52431, December 26, 2003

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Differential Processing of CD4 T-cell Epitopes from the Protective Antigen of Bacillus anthracis^*

Julie A. Musson, Nicola Walker, Helen Flick-Smith, E. Diane Williamson, and John H. Robinson¶

From the Musculoskeletal Research Group, Clinical Medical Sciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH and the Defense Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom

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.

Received for publication, August 14, 2003 , and in revised form, October 6, 2003.

^* This work was funded in part by a Defense Science and Technology Laboratory extramural research contract. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 44-0191-222-6976; Fax: 44-0191-222-5455; E-mail: j.h.robinson{at}ncl.ac.uk.

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