Differential Processing of CD4 T-cell Epitopes from the Protective Antigen of Bacillus anthracis (2003)

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. 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. The protective antigen of Bacillus anthracis (PA) 1The abbreviations used are: PAprotective antigen of B. anthracisrPArecombinant protective antigenAPCantigen presenting cellMHCmajor histocompatibility complexE-64d(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl esterDCI3,4-dichloroisocoumarinTPCKN-p-Tosyl-l-phenylalanine chloromethyl ketoneTLCKNα-p-Tosyl-l-lysine-chloromethyl ketone hydrochloride. binds to a host cell-surface receptor before cleavage and heptamerization to form a transmembrane pore that delivers two further toxin subunits, edema and lethal factors, into the host cell cytosol (1Petosa C. Liddington R.C. Parker M.W. Protein Toxin Structure. Landes, Georgetown1996: 97-119Crossref Google Scholar, 2Beauregard K.E. Collier R.J. Swanson J.A. Cell. Microbiol. 2000; 2: 251-258Crossref PubMed Scopus (92) Google Scholar, 3Sellman B.R. Mourez M. Collier R.J. Science. 2001; 292: 695-697Crossref PubMed Scopus (166) Google Scholar, 4Sellman B.R. Nassi S. Collier R.J. 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A. 2002; 99: 7049-7053Crossref PubMed Scopus (151) Google Scholar, 10Lacy D.B. Mourez M. Fouassier A. Collier R.J. J. Biol. Chem. 2002; 277: 3006-3010Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) in relation to the four domains of the PA molecule. In addition to a role in virulence, PA is the main target of protective immunity to anthrax and is thought to induce T-cell-dependent antibodies that protect the host by preventing toxin assembly and internalization (11Coulson N.M. Fulop M. Titball R.W. Vaccine. 1994; 12: 1395-1401Crossref PubMed Scopus (74) Google Scholar, 12McBride B.W. Mogg A. Telfer J.L. Lever M.S. Miller J. Turnbull P.C. Baillie L. Vaccine. 1998; 16: 810-817Crossref PubMed Scopus (97) Google Scholar, 13Singh Y. Ivins B.E. Leppla S.H. Infect. Immun. 1998; 66: 3447-3448Crossref PubMed Google Scholar, 14Barnard J.P. Friedlander A.M. Infect. Immun. 1999; 67: 562-567Crossref PubMed Google Scholar, 15Baillie L. J. Appl. Microbiol. 2001; 91: 609-613Crossref PubMed Scopus (50) Google Scholar). Consequently, PA is the major antigenic component of the current anthrax vaccine, and subunit vaccines based on truncated recombinant PA molecules are under development (16Flick-Smith H.C. Walker N.J. Gibson P. Bullifent H. Hayward S. Miller J. Titball R.W. Williamson E.D. Infect. Immun. 2002; 70: 1653-1656Crossref PubMed Scopus (103) Google Scholar). However, the relationship between the assembly and function of PA as a toxin-translocating pore and the mechanisms of antigen processing and presentation of PA to CD4+ T-cells has not been elucidated. protective antigen of B. anthracis recombinant protective antigen antigen presenting cell major histocompatibility complex (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester 3,4-dichloroisocoumarin N-p-Tosyl-l-phenylalanine chloromethyl ketone Nα-p-Tosyl-l-lysine-chloromethyl ketone hydrochloride. Peptide epitopes recognized by CD4+ T-cells are liberated from protein antigens following some form of antigen processing before they are accessible to bind MHC class II molecules usually in acidic endocytic compartments of antigen presenting cells (APC) such as macrophages, dendritic cells, and B cells (17Nakagawa T.Y. Rudensky A.Y. Immunol. Rev. 1999; 172: 121-129Crossref PubMed Scopus (161) Google Scholar, 18Riese R.J. Chapman H.A. Curr. Opin. Immunol. 1999; 12: 107-113Crossref Scopus (181) Google Scholar, 19Pieters J. Adv. Immunol. 2000; 75: 159-208Crossref PubMed Google Scholar, 20Villadangos J.A. Ploegh H.L. Immunity. 2000; 12: 233-239Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 21Watts C. Curr. Opin. Immunol. 2001; 13: 26-31Crossref PubMed Scopus (131) Google Scholar, 22Lennon-Duménil A.-M. Bakker A.H. Wolf-Bryant P. Ploegh H.L. Lagaudrière-Gesbert C. Curr. Opin. Immunol. 2002; 14: 15-21Crossref PubMed Scopus (119) Google Scholar, 23Unanue E.R. Immunol. Rev. 2002; 185: 86-102Crossref PubMed Scopus (78) Google Scholar, 24Robinson J.H. Delvig A.A. Immunology. 2002; 105: 252-262Crossref PubMed Scopus (80) Google Scholar). Following uptake by APC, processing is thought to be initiated during transport through the endosomal system by unfolding of antigens induced by low pH (25Eisenlohr L.C. Gerhard W. Hackett C.J. J. Immunol. 1988; 141: 1870-1876PubMed Google Scholar, 26Jensen P.E. J. Immunol. 1993; 150: 3347-3356PubMed Google Scholar), oxidation (27Carrasco-Marín E. Paz-Miguel J.E. López-Mato P. Alvarez-Domínguez C. Leyva-Cobián P. Immunology. 1998; 95: 314-321Crossref PubMed Scopus (43) Google Scholar), disulphide reduction (28Jensen P.E. Semin. Immunol. 1995; 7: 347-353Crossref PubMed Scopus (35) Google Scholar, 29Quin S.J. Seixas E.M.G. Cross C.A. Berg M. Lindo V. Stockinger B. Langhorne J. Eur. J. Immunol. 2001; 31: 72-81Crossref PubMed Scopus (36) Google Scholar, 30Maric M. Arunachalam B. Phan U.T. Dong C. Garrett W.S. Cannon K.S. Alfonso C. Karlsson L. Flavell R.A. Cresswell P. Science. 2001; 294: 1361-1365Crossref PubMed Scopus (221) Google Scholar), or limited cleavage by lysosomal enzymes such as asparaginyl endopeptidase (31Antoniou A.N. Blackwood S.-L. Mazzeo D. Watts C. Immunity. 2000; 12: 391-398Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Some or all of these steps may be essential to “unlock” or progressively expose protein antigens to further proteolytic degradation by lysosomal hydrolases, including cathepsins of the cysteine and aspartyl enzyme families (18Riese R.J. Chapman H.A. Curr. Opin. Immunol. 1999; 12: 107-113Crossref Scopus (181) Google Scholar, 22Lennon-Duménil A.-M. Bakker A.H. Wolf-Bryant P. Ploegh H.L. Lagaudrière-Gesbert C. Curr. Opin. Immunol. 2002; 14: 15-21Crossref PubMed Scopus (119) Google Scholar). Serine proteinases have been implicated in antigen processing in the extracellular space for presentation of peptide epitopes on the surface of dendritic cells (32Accapezzato D. Nisini R. Paroli M. Bruno G. Bonino F. Houghton M. Barnaba V. J. Immunol. 1998; 160: 5262-5266PubMed Google Scholar, 33Santambrogio N. Sato A.K. Carven G.J. Belyanskaya S.L. Strominger J.L. Stern L.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15056-15061Crossref PubMed Scopus (169) Google Scholar), but the role of serine proteinases in intracellular processing has not been studied in any detail (34Takahashi H. Cease K.B. Berzofsky J.A. J. Immunol. 1989; 142: 2221-2229PubMed Google Scholar, 35Puri J. Factorovich Y. J. Immunol. 1988; 141: 3313-3317PubMed Google Scholar, 36Delvig A.A. Robinson J.H. J. Biol. Chem. 1998; 273: 3291-3295Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Peptide epitopes liberated during processing usually bind newly synthesized MHC class II molecules optimally at low pH under the control of chaperones DM and DO following degradation of the invariant chain. Alternatively, epitopes in structurally flexible regions of protein antigens may bind mature MHC class II recycled from the APC surface in neutral or mildly acidic endosomal compartments (24Robinson J.H. Delvig A.A. Immunology. 2002; 105: 252-262Crossref PubMed Scopus (80) Google Scholar, 37Sinnathamby G. Eisenlohr L.C. J. Immunol. 2003; 170: 3504-3513Crossref PubMed Scopus (47) Google Scholar). Further processing or “trimming” of exposed amino acids of the peptides already bound to MHC molecules can occur (38Sercarz E.E. Maverakis E. Nat. Rev. Immunol. 2003; 3: 621-629Crossref PubMed Scopus (136) Google Scholar), mediated by enzymes such as the metalloproteinase aminopeptidase N (39Amoscato A.A. Prenovitz D.A. Lotze M.T. J. Immunol. 1998; 161: 4023-4032PubMed Google Scholar). Peptide-MHC complexes are transported to the APC surface where they can bind T-cell antigen receptors in the formation or an immunological synapse leading to T-cell activation of anergy (40van der Merwe P.A. Davis S.J. Ann. Rev. Immunol. 2003; 21: 659-684Crossref PubMed Scopus (433) Google Scholar). We investigated how recombinant PA is processed by macrophages for presentation to CD4+ T-cell hybridomas and conclude that T-cell epitopes of PA may be sequentially for presentation at stages of endosomal processing. and PA protein of B. anthracis was and in and as (16Flick-Smith H.C. Walker N.J. Gibson P. Bullifent H. Hayward S. Miller J. Titball R.W. Williamson E.D. Infect. Immun. 2002; 70: 1653-1656Crossref PubMed Scopus (103) Google Scholar). peptides amino of the PA were synthesized by the The of PA structure in were by the Protein from the and and inhibitors were from cells were in and T-cell hybridomas were generated by of cells by P. of and and of with CD4+ T-cell from and The of T-cell hybridomas was from their to peptides the PA T-cell specific for of the major epitopes in domains and of recognized as as peptides including the of the epitope presented by of antigen G. C. P. R. F. L. J. PubMed Scopus Google Robinson C.J. N.J. J. Chem. Chem. Google K. PubMed Scopus Google M. D. F. H. J. A. J. Immunol. PubMed Scopus Google M. D. F. H. J. A. J. Immunol. PubMed Scopus Google M. D. F. H. J. A. J. Immunol. PubMed Scopus Google M. K. S. Y. K. J. PubMed Scopus Google J. Factorovich Y. J. Immunol. 1988; 141: 3313-3317PubMed Google H. Cease K.B. Berzofsky J.A. J. Immunol. 1989; 142: 2221-2229PubMed Google in a of T-cell hybridomas The from T-cell responses to amino peptides are in were in the of the in a marrow-derived macrophages were used as the of were from bone cells of and by for in with of a from the cell as a of as B. K. Eur. J. Immunol. 1988; PubMed Scopus Google Scholar). were by treatment with before in antigen presentation were to to for at in a were with inhibitors for min before antigen in the form of or peptides for to and were to in for and was with followed by three The of macrophages was by in all the inhibitors were not for cells in the T-cell cells were to the macrophages, and were for before macrophages were used as The of T-cell hybridomas was as the of in a as of cells in the of T-cell and for at in of The cells were with of specific activity for and was a and are as of were at and the data for are of of PA by mapped epitopes in domains and of PA and generated T-cell hybridomas specific for epitope and T-cell hybridomas were for their to presented by bone macrophages to study the mechanisms of antigen processing of before addition of to induce responses in any of the T-cell hybridomas not that all epitopes required uptake processing of the intact for presentation to were with for before and addition of T-cell hybridomas to the kinetics of presentation of epitope The epitope was already presented optimally only 15 min of to before of macrophages. The responses to epitopes and more and were by to of peptides epitope and was more in kinetics with suggesting that the of responses to was not a of in the of peptide epitopes for MHC class II or the T-cell receptor of the Thus, the data that PA epitopes were processed and presented to T-cells at of in of of antigens from to the low pH of or is required for presentation of (17Nakagawa T.Y. Rudensky A.Y. Immunol. Rev. 1999; 172: 121-129Crossref PubMed Scopus (161) Google Scholar) but not all (24Robinson J.H. Delvig A.A. Immunology. 2002; 105: 252-262Crossref PubMed Scopus (80) Google Scholar, A.A. Robinson J.H. J. Immunol. 1998; 160: Google Scholar, J.A. Hayward Delvig A.A. R. V. Robinson J.H. Eur. J. Immunol. 2002; PubMed Scopus Google Scholar) CD4+ T-cell were with a that endosomal pH R. A. Ann. Rev. PubMed Google Scholar) to whether a low pH is required for presentation of of the PA of epitopes and was unaffected by treatment of APC with ammonium chloride and presentation of and was independent of antigen and chloride not the of macrophages to peptides epitope The data that presentation of T-cell epitopes of showed a on low pH of of and on of families of enzymes are in processing of T-cell epitopes from investigated the of treatment of macrophages with broad spectrum proteinase inhibitors. The enzymes to be required for antigen presentation are lysosomal cathepsins of the cysteine and aspartic proteinase families that proteolytic processing of the protein antigen or the MHC class II invariant C. Curr. Opin. Immunol. 2001; 13: 26-31Crossref PubMed Scopus (131) Google Scholar, 22Lennon-Duménil A.-M. Bakker A.H. Wolf-Bryant P. Ploegh H.L. Lagaudrière-Gesbert C. Curr. Opin. Immunol. 2002; 14: 15-21Crossref PubMed Scopus (119) Google Scholar). We used broad spectrum inhibitors of these families to whether enzymes to were required for presentation of PA ethyl is a that cysteine proteinases, including cathepsins and B P.E. Ann. Rev. PubMed Scopus Google Scholar, M. K. S. Y. K. J. PubMed Scopus Google Scholar). presentation of and at all However, presentation of was more whereas epitopes and were unaffected by treatment is an used to aspartic proteinases including cathepsins and J. Factorovich Y. J. Immunol. 1988; 141: 3313-3317PubMed Google Scholar). of was to treatment of macrophages with whereas the epitopes were to Thus, the presented epitope to be independent of processing by lysosomal cysteine and aspartic the more presented epitopes the two from of PA and showed a on cysteine proteinase whereas the epitope of was the by of aspartic of Serine and on of investigated the role of serine proteinases to whether they were required for processing and presentation of PA were with the broad spectrum serine proteinase before their in antigen presentation presentation of all epitopes under study from a of antigen the responses to peptides the were macrophages with broad spectrum serine proteinase not The more serine proteinase inhibitors and were used to and serine proteinases, presentation of all epitopes to the a of a with and a and and at a and on peptide The data that presentation of required serine proteinase activity but that epitopes showed of on serine proteinase of macrophages with the broad spectrum metalloproteinase a on presentation of and whereas presentation of was unaffected and a of of presentation of was suggesting that metalloproteinases are during processing of to presentation of some epitopes, but of We mapped epitopes in of the domains of the anthrax protective antigen to study proteolytic processing for MHC class antigen presentation by macrophages. We used macrophages as APC and with inhibitors to the mechanisms of proteolytic processing for presentation to T-cell hybridomas specific for of the epitopes epitopes located in and showed a of processing in the to inhibitors of cysteine and serine proteinases, as as on metalloproteinase activity and to raising endosomal is not accessible on the surface of whereas is located on a to the cleavage However, epitopes to require a of processing for the two epitopes in presentation of showed a on and metalloproteinase but was to raising endosomal the suggesting processing by of these enzyme families at neutral or pH K. E. Biol. Chem. 2002; PubMed Scopus Google Scholar). In contrast, presentation of located in to the of was but unaffected by inhibitors of cysteine and the on aspartic proteinase Thus, these four epitopes of PA showed for proteolytic processing. In particular, and showed the that these epitopes are located in in suggesting the of an epitope is a major in the is liberated for MHC binding and from showed the of processing based on the on low and to treatment of macrophages with inhibitors of and suggesting that lysosomal processing was not In the presentation of by macrophages with that metalloproteinases was not presented from the intact by macrophages not and presentation was dependent on the activity of serine proteinases, suggesting that following limited proteolytic cleavage of at an before transport the endosomal of and the of to raising endosomal We have that epitopes were presented by mature MHC class II A. and J. H. suggesting presentation of and in by MHC class II in the In contrast, presentation of and was dependent on low and have that these epitopes were presented by newly synthesized MHC class II of to acidic intracellular compartments for presentation by the enzymes such as the cysteine proteinase have a role in degradation of invariant is required for peptide of newly synthesized MHC class II molecules J.A. R.J. C. Chapman H.A. Ploegh H.L. J. 1997; PubMed Scopus Google Scholar). of PA epitopes, to cysteine proteinase inhibitors may not a role for enzymes such as in cleavage of but is with processing for the A.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and dendritic cells A. J. Leppla S.H. S. A. C. B. Nature. 2003; PubMed Scopus Google Scholar) are the of anthrax lethal toxin PA binds to a cell surface receptor (8Bradley K.A. Mogridge J. Mourez M. Collier R.J. Young J.A.T. Nature. 2001; 414: 225-229Crossref PubMed Scopus (757) Google Scholar) followed by cleavage into and by cell serine proteinases, P.A. Leppla S.H. Klimpel K.R. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). cleavage of PA to heptamerization of the to form a the by S.H. J. B. M. and in Scholar). The epitopes in are located within the and their presentation was dependent on serine proteinase activity consistent with a requirement for in a study of the mechanisms of uptake of for presentation to CD4+ a specific presentation of and but not in other domains of A. N. H. E. D. and J. H. for Thus, cleavage may be required to for presentation of epitopes and but not for uptake of PA for presentation of the PA The kinetics of presentation of from with the other epitopes of PA studied that epitopes are liberated from at The requirement for serine proteinase activity independent of low pH for presentation of processing of by neutral proteinases in or at the cell surface during uptake of The majority of serine proteinases have pH and at low such as of the F. R.A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), the endopeptidase activity for cleavage within protein antigens required for antigen of the epitopes of PA required serine proteinase but in these was in addition to the activity of or The cysteine and aspartic proteinase activity is to be of lysosomal but is not the for the metalloproteinase as some of enzyme have pH such as aminopeptidase N located at the (39Amoscato A.A. Prenovitz D.A. Lotze M.T. J. Immunol. 1998; 161: 4023-4032PubMed Google Scholar). Thus, metalloproteinases in prior to lysosomal processing during antigen the data that processing of sequentially in a of steps beginning with cleavage by neutral proteinases before with lysosomal enzymes. epitopes such as step is for presentation to occur is consistent with the exposed location of on the surface of of However, for the majority of epitopes processing may be initiated by neutral proteinases followed by further cleavage during lysosomal processing and of MHC class Serine proteinases have been implicated in or extracellular antigen processing for MHC class II molecules at the cell surface (32Accapezzato D. Nisini R. Paroli M. Bruno G. Bonino F. Houghton M. Barnaba V. J. Immunol. 1998; 160: 5262-5266PubMed Google Scholar, 33Santambrogio N. Sato A.K. Carven G.J. Belyanskaya S.L. Strominger J.L. Stern L.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15056-15061Crossref PubMed Scopus (169) Google Scholar, R. S. P. J. Immunol. Google Scholar), but their in endosomal processing within APC has not been studied in of APC with some inhibitors of serine proteinase activity was to have a limited on presentation of epitopes of (34Takahashi H. Cease K.B. Berzofsky J.A. J. Immunol. 1989; 142: 2221-2229PubMed Google Scholar) or J. Factorovich Y. J. Immunol. 1988; 141: 3313-3317PubMed Google Scholar). However, have of presentation of T-cell epitopes of the surface protein of to the chloromethyl and A.A. Robinson J.H. J. Biol. Chem. 1998; 273: 3291-3295Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), and have used the more and broad spectrum serine proteinase to enzyme in antigen processing. to before further to epitopes for presentation have been but the are to lysosomal enzymes. the enzyme lysosomal antigen presentation of MHC class T-cell responses to a of protein antigens M. Arunachalam B. Phan U.T. Dong C. Garrett W.S. Cannon K.S. Alfonso C. Karlsson L. Flavell R.A. Cresswell P. Science. 2001; 294: 1361-1365Crossref PubMed Scopus (221) Google Scholar). of during antigen processing was to be a step in the availability of T-cell epitopes within the surface of S.J. Seixas E.M.G. Cross C.A. Berg M. Lindo V. Stockinger B. Langhorne J. Eur. J. Immunol. 2001; 31: 72-81Crossref PubMed Scopus (36) Google Scholar). However, the of enzyme may not be an of antigen presentation, that the amino of PA not any cysteine pre-processing role has been for the lysosomal cysteine proteinase asparaginyl enzyme was to an in the of toxin essential to a of CD4+ T-cell epitopes of their to the cleavage (31Antoniou A.N. Blackwood S.-L. Mazzeo D. Watts C. Immunity. 2000; 12: 391-398Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), suggesting that a cleavage of the intact protein to extensive unfolding and of cleavage sites for processing enzymes. We that pre-processing at an earlier in the endocytic mediated by serine proteinases, an enzyme that in the extensive antigen presentation We are the of the serine proteinase activity in antigen processing by with compartments of macrophages by J.P. R. J. Immunol. 1997; Google Scholar, A.A. L. Robinson J.H. Eur. J. Immunol. 2003; PubMed Scopus Google Scholar). We have that PA with to and that can be presented to a T-cell by macrophages. activity is to of the serine proteinase during suggesting cleavage of by serine proteinases is for presentation of clearer of the of that to processing and presentation of protective antigens such as PA may be an essential step antigens to induce responses that a to We for of the