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*
Unité de Biologie des Régulations Immunitaires and
Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, it has been well established that in some cases, exogenous Ag can be processed and presented to CD8+ T cells (reviewed in Refs. 2–4). Schematically, these pathways have been classified as either cytosolic or noncytosolic. Noncytosolic pathways are related to the endocytic processing and involve loading of peptides on post-Golgi MHC class I molecules and are independent of the TAP system 2, 3 . Cytosolic pathways constitute a nonconventional access of exogenous Ag to the cytosol, resulting in its introduction into the classic presentation pathway for endogenous Ag described above. This has been clearly demonstrated for particulate Ag internalized by phagocytosis in a restricted subset of macrophages 5, 6, 7 and for soluble Ag internalized by macropinocytosis in both activated macrophages 8 and dendritic cells 9, 10 . In both cases, a defined endocytic mechanism (phagocytosis or macropinocytosis) was involved, and a membrane-disruption mechanism was suggested to explain the introduction of exogenous Ag into the cytosolic pathway.
Several bacterial toxins exert their toxic effect by modifying cytosolic components. Therefore, they are equipped with a dedicated translocating capability, and as such, they constitute attractive vectors for the delivery of heterologous CTL epitopes into the endogenous pathway. Some toxins, such as the diphtheria toxin, are internalized after receptor-mediated endocytosis in an acidic compartment where they translocate to the cytosol 11 . Other toxins use a receptor-mediated anterograde transport to the ER via the Golgi network, where they translocate to the cytosol (reviewed in 12 . The adenylate cyclase toxin of Bordetella pertussis (CyaA) is able to invade numerous mammalian cell types, in which, upon activation by the endogenous calmodulin, it catalyzes the production of supraphysiological levels of cAMP 13, 14, 15, 16, 17, 18, 19 . The invasivity of CyaA seems to be independent of any known cellular receptor: it does not involve any acidic compartment and can take place in erythrocytes devoid of vesicular transport 20, 21, 22, 23, 24, 25, 26, 27 . The precise mechanism of translocation is not fully understood but might occur in two steps. After binding of CyaA to the eukaryotic cell surface mediated by the C-terminal domain (residues 400-1706), the N-terminal catalytic domain (residues 1–400) is directly translocated through the plasma membrane into the cell.
We showed previously that a CyaA toxin genetically tagged with a CTL epitope from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) into its catalytic domain remained cell invasive 28 . Moreover, this toxin was able to generate, in vivo, a protective CTL response against LCMV 29, 30 . In this report, we asked whether the CyaA toxin, taken as an exogenous Ag, can be delivered to the cytosolic pathway for MHC class I presentation without the contribution of any endocytic mechanisms. To address this question, we have analyzed, in vitro, the pathway leading to the presentation of the Kb-restricted CD8+ OVA epitope from OVA, which was inserted in a genetically detoxified form of CyaA (CyaA-OVA E5). We used the stimulation of a specific CD8+ T cell hybridoma to monitor the appearance of Kb/OVA complexes on the surface of APC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) mice from Iffa Credo (L’Arbresle, France) were used between 6 and 8 wk of age. Female TAP1 knockout mice 31 bred onto a C57BL/6 background were a kind gift from Dr. A. Bandeira (Institut Pasteur, Paris, France).
Recombinant adenylate cyclase toxins and peptide
The synthetic peptide SIINFEKL corresponding to the CD8+ T cell epitope encompassing the OVA residues 257–264 recognized by the B3Z CD8+ T cell hybridoma was synthesized by Neosystem (Strasbourg, France) and stored in PBS.
The wild-type CyaA toxin was produced in Escherichia coli from an expression plasmid, pCACT3, which carries the cya C gene required for the activation of the pro-CyaA toxin and the modified cya A gene under the transcriptional control of the lac UV5 promoter 28 .
CyaA-OVA E5 is a genetically detoxified CyaA toxin carrying the OVA epitope. It differs from the wild-type CyaA toxin by 1) the insertion between Arg224 and Ala225 of the amino acid sequence, PASIINFEKLGT (the amino acid sequence of the OVA epitope is underlined), and 2) the insertion between Asp188 and Ile189 of the dipeptide LQ. This latter modification abolishes the adenylate cyclase activity without affecting the cell-invasive activity 32 . Details of the construction can be provided upon request. The genetically detoxified CyaA toxin carrying the LCMV CTL epitope (CyaA-LCMV E5) (due to the same LQ insertion between Asp188 and Ile189) carrying the 118–132 CD8+ T cell (H-2d restricted) epitope from the LCMV nucleoprotein has been previously described 29 . The CyaA toxins, overproduced in the E. coli BLR strain, accumulated as inclusion bodies. After solubilization in 8 M urea, 20 mM HEPES-sodium (pH 7.5) they were purified to >95% homogeneity (as judged by SDS-gel analysis, not shown) by two sequential chromatographies on DEAE-Sepharose and phenyl-Sepharose. Toxin concentrations were determined spectrophotometrically from the absorption at 280 nm using a molecular extinction coefficient of 142,000 M-1 · cm-1.
Cell lines
B3Z 33 , a CD8+ T cell hybridoma specific for the OVA 257–264 peptide (SIINFEKL) in the context of Kb, was a generous gift from Dr. N. Shastri (University of California, Berkeley, CA). The LB27.4 B cell lymphoma and the EL4 thymoma were obtained from American Type Culture Collection (Manassas, VA). The immortalized immature dendritic cell line FSDC derived from fetal skin 34 was a kind gift from Dr. P. Ricciardi-Castagnoli (Consiglio Nazionale delle Ricerche, Milan, Italy).
Preparation of thioglycollate-induced peritoneal exudate cells (Thio-PEC), bone marrow macrophages (BMM), enriched populations of bone marrow dendritic cells (BMDC), and splenocytes
C57BL/6 mice were injected i.p. with 2 ml of thioglycollate (Sanofi Diagnostic Pasteur, Paris, France). Four to seven days later, mice were sacrificed and the cells infiltrating the peritoneal cavity were harvested by washing with 10–15 ml of RPMI 1640 containing antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). The cells were then pelleted, resuspended in complete medium (CM, consisting of RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 5 x 10-5 M 2-ME), and used for presentation experiments.
BMM were obtained after a 5-day culture of bone marrow cells harvested on naive C57BL/6 mouse femurs in CM supplemented with 10% of macrophage CSF (M-CSF) containing supernatant of L929 cell culture. Cultures were realized in bacteriology petri dishes (Becton Dickinson, Mountain View, CA). At the end of the culture period, nonadherent cells were removed, and adherent cells were harvested with 2 mM trypsin-EDTA (NEN Life Science, Boston, MA).
BMDC were obtained with a protocol adapted from Inaba et al. 35 . Briefly, bone marrow cells were washed twice and cultured for 2 days in DC medium (CM supplemented with recombinant granulocyte-macrophage CSF (GM-CSF; Peprotech, Rocky Hill, NJ) at 40 ng/ml) at 106 cells/ml in 100-mm culture-treated petri dishes. Then, nonadherent cells were removed, and fresh DC medium was added. After a total of 5–6 days of culture, BMDC were visible as clumps of loosely adherent cells. The nonadherent cells were harvested by vigorous pipetting, washed, and used for presentation experiments.
For the preparation of splenocytes, spleen cells from naive C57BL/6 mice were washed once with RPMI 1640 supplemented with antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), resuspended in CM, and used for presentation assays.
Ag presentation assays
The stimulation of B3Z cells (105 cells/well) was
monitored by IL-2 release in the supernatants of 24-h cultures in the
presence of APC in 96-well culture plates. In some experiments (see
legends to Figs. 1
and 7), the APC (105 cells/well
except for FSDC, 104 cells/well) were cocultured with B3Z
in the presence of Ag. In other experiments, APC were first pulsed with
Ag for 5 h and then fixed with glutaraldehyde 0.05% (2 min at
37°C) and washed twice before being cocultured with B3Z. The B3Z
stimulation was done in 96-well culture microplates in a 0.2-ml final
volume in the presence of 0.25 µM indomethacin (Sigma, St. Louis,
MO). The Ag concentrations used in each experiment are indicated in the
figure legends. After 24 h, the supernatants were harvested and
frozen for at least 2 h at -70°C. Then, 104
cells/well of the CTLL cell line, which proliferates specifically in
response to IL-2, were cultured with 100 µl of supernatant in 0.2-ml
final volume. Two days later, [3H]thymidine (NEN Life
Science) was added, and the cells were harvested 18 h later with
an automated cell harvester (Skatron, Lier, Norway). Incorporated
thymidine was detected by scintillation counting. In all experiments,
each point was done at least in duplicate and more often in triplicate.
Results are expressed in cpm or 
cpm (cpm in the presence of Ag
- cpm in the absence of Ag).
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For inhibition studies, Thio-PEC APC were first incubated in 0.1 ml with the drugs for 1 h at the indicated final concentration. Then, Ag diluted in 0.1 ml were added to the wells (0.2-ml final volume) at the final concentration indicated, in the continuous presence of the inhibitor for 5 h. APC were then fixed with glutaraldehyde 0.05% (2 min at 37°C) and washed twice, and the B3Z T cells were added to the wells and cultured for 18 h in a 0.2-ml final volume as indicated above. Lactacystin (Tebu, Le Perray-en-Yvelines, France) was dissolved in PBS at 1 mg/ml, brefeldin A (BFA; Sigma) was dissolved in methanol at 0.5 mg/ml, dimethyl amiloride (DMA, Sigma) was dissolved in DMSO at 10 mM, and cytochalasine B (CCB) and N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL, Sigma) were dissolved in DMSO at 10 mg/ml. Cycloheximide (Sigma) was dissolved in water at 2 mg/ml. Chloroquine (Sigma) was dissolved in CM at 10 mM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using thioglycollate-induced peritoneal macrophages (Thio-PEC)
from C57BL/6 (H-2b) mice as APC, we monitored the formation
of Kb-SIINFEKL complexes by the stimulation of B3Z, a
CD8+ T cell hybridoma specific for the 257–264 SIINFEKL
peptide of chicken OVA 33 . Fig. 1
shows
that B3Z cells cocultured for 18 h with Thio-PEC were efficiently
stimulated in the presence of CyaA-OVA E5, a genetically inactivated
CyaA toxin that carries the SIINFEKL epitope. In contrast, a detoxified
toxin carrying an irrelevant epitope (CyaA-LCMV E5) or the
nonrecombinant CyaA was unable to stimulate the B3Z T cells. As
expected, the stimulation of B3Z was specifically blocked by an
anti-Kb mAb and was not observed with MHC-mismatched
Thio-PEC as APC (data not shown), confirming that stimulation was due
to the specific recognition of Kb/SIINFEKL complexes.
Moreover, the presentation of CyaA-OVA E5 required processing, since
the stimulation of B3Z by CyaA-OVA E5 was observed only with live APC
but not with prefixed APC (data not shown).
CyaA-OVA E5 processing requires proteasome activity
We next characterized the degradation steps involved in the
generation of the SIINFEKL CD8+ T cell epitope from the
recombinant CyaA-OVA E5. It is now well established that the processing
of cytosolic Ag for loading on MHC class I molecules requires the
proteolytic activities of the proteasome 36, 37, 38 . As shown in Fig. 2, LLnL and lactacystin, two inhibitors
of the proteasome activity 37, 38 , totally inhibited the presentation
of CyaA-OVA E5 (Fig. 2A) without altering the presentation
of the SIINFEKL synthetic peptide (Fig. 2B). We conclude
that the proteolytic processing of CyaA-OVA E5 to yield the SIINFEKL
peptide is dependent on the proteasome function.
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The intracytoplasmic location of the proteasome-mediated
degradation of CyaA-OVA E5 raised the question of the translocation of
the processed peptides toward a compartment containing MHC class I
molecules. To test the intervention of the TAP system in the
presentation pathway of CyaA-OVA E5, we compared the presentation of
this Ag by Thio-PEC obtained from C57BL/6 wild-type mice (+/+) and from
C57BL/6 TAP1 knockout mice (-/-), which do not have a functional TAP
system 31 . Fig. 3 shows that both
APC populations have a similar ability to present the SIINFEKL
synthetic peptide to B3Z, whereas only APC from wild-type mice
presented CyaA-OVA E5 to B3Z. Thus, it can be concluded that the TAP
system is required for the transport of the processed OVA peptide from
the cytosol to the lumen of the ER.
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The TAP requirement for the presentation of CyaA-OVA E5 to B3Z
suggests that the SIINFEKL peptide generated from the recombinant toxin
might be loaded on newly synthesized MHC class I molecules in the lumen
of the ER. To test this possibility, we disrupted the conventional
secretory pathway with the drug BFA, which has been shown to inhibit
the export of newly synthesized MHC class I-peptide complexes from the
ER to the cellular membrane 39 . BFA completely inhibited the
presentation of CyaA-OVA E5 to B3Z (Fig. 4A), whereas it did not modify
the presentation of the SIINFEKL synthetic peptide (Fig. 4B). Cycloheximide, a potent inhibitor of protein synthesis,
had a similar effect (Fig. 4C). These results indicate that
the processed peptides bind to nascent MHC class I molecules in the
lumen of the ER and that the complexes formed are exported to the cell
surface via the Golgi apparatus by the conventional secretory pathway.
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The presentation of CyaA-OVA E5 requires extracellular Ca2+
The translocation of the CyaA catalytic domain through the plasma
membrane of eukaryotic cells is critically dependent upon extracellular
Ca2+ 20 . To test whether the CyaA-OVA E5 presentation by
Thio-PEC is mediated by a Ca2+-dependent internalization
mechanism, we performed experiments in the presence of EGTA to deplete
extracellular Ca2+ (Fig. 5).
EGTA was used in a 15-min Ag pulse followed by a 5-h chase in the
absence of EGTA and Ag (Fig. 5). Under these conditions, EGTA
completely inhibited the CyaA-OVA E5 presentation without affecting the
presentation of the SIINFEKL synthetic peptide. This suggests that the
Ca2+-dependent direct translocation of the catalytic domain
of CyaA-OVA E5 into APC is the prerequisite for presentation.
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The internalization of CyaA has already been shown to be
independent of vacuolar acidification 23 . However, it would be
possible that an endocytic acid-optimal proteolysis event could
participate in the processing of CyaA-OVA E5 to give rise to the
SIINFEKL epitope. To investigate this possibility, we tested the effect
of the weak base chloroquine on the presentation of CyaA-OVA E5. As
shown in Fig. 6, chloroquine that
inhibits the endolysosomal proteolysis and interferes with vesicular
transport affected neither the presentation of CyaA-OVA E5 nor the
presentation of the SIINFEKL synthetic peptide. EGTA was included in
this experiment as a negative control.
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Macropinocytosis is a recently described form of endocytosis distinct
from the clathrin-mediated endocytosis associated with
membrane-ruffling activity. The uptake of soluble Ag by
macropinocytosis can result in delivery of the Ags to the
cytosol and their subsequent introduction in the endogenous pathway for
MHC class I-restricted Ag presentation. Macropinocytosis has been shown
to be inducible in macrophages and constitutive in dendritic cells
8, 9, 10 . To determine whether the delivery of CyaA-OVA E5 in the
cytosolic pathway was related to the macropinocytic route, we tested
the effect of DMA, a potent inhibitor of the macropinocytic pathway, on
the presentation of CyaA-OVA E5. As shown in Fig. 6, DMA was without
effect on CyaA-OVA E5 presentation. This demonstrates that the CyaA-OVA
E5 pathway represents an original mechanism for the presentation of
exogenous Ag to CD8+ T cells through the direct delivery of
epitope into the cytosol through the plasma membrane of APC.
CyaA-OVA E5 presentation by various types of APC
Previously described pathways for the presentation of exogenous Ag
by MHC-class I molecules have been shown to be restricted to defined
cell types, mainly macrophages and dendritic cells. For example,
phagocytic and macropinocytic pathways clearly exhibit a cell-type
specificity linked to the endocytic mechanism underlying the access of
Ag to the cytoplasm. As CyaA is able to enter into various eukaryotic
cell types independently of receptor-mediated endocytosis 20, 22, 23, 24, 25, 26, 27 ,
processing and presentation of CyaA-OVA E5 should be conducted by
different cell types, the possible consequence being that the
presentation of CyaA-OVA E5 might take place in multiple cell types. To
test this hypothesis, we examined the presentation of CyaA-OVA E5 by
different types of APC. Macrophages derived from bone marrow by culture
with M-CSF presented CyaA-OVA E5 with a slightly reduced efficiency
compared with macrophages derived from the peritoneal cavity after
thioglycollate injection (Thio-PEC). Dendritic cells derived from bone
marrow by culture in GM-CSF were able to present CyaA-OVA E5 with an
efficiency comparable with that of Thio-PEC (Fig. 7A). To exclude the
possibility that granulocytes contaminating the BMDC preparation
contributed to the presentation of CyaA-OVA E5, we performed
experiments with the FSDC dendritic cell line. The FSDC cell line was
able to present CyaA-OVA E5 to B3Z (Fig. 7, A and
B). The weak efficiency of presentation is probably linked
to the immature phenotype of this cell line. Unfractionated
splenocytes, as well as the EL4 thymoma and the LBZ7.4 B cell lymphoma,
were also able to present the CyaA-OVA E5 to B3Z T cells (Fig. 7C).
Thus, taken together, our results establish that, despite differences in efficiency, several APC of both myeloid and lymphoid origin were able to present the CyaA-OVA E5 to B3Z CD8+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The use of recombinant toxins for the delivery into the cytosol of CTL epitopes gives rise to an increasing interest. Besides CyaA, two other systems have recently been developed using the Pseudomonas aeruginosa exotoxin 41, 42 and the lethal factor (LF) of Bacillus anthracis in the presence of the protective Ag component of the toxin 43, 44, 45 . In both cases, sensibilization of target cells was effective in vitro, but the recombinant LF of B. anthracis was also shown to elicit a CTL response in vivo against the inserted epitope 44 .
The presentation of the recombinant P. aeruginosa exotoxin toxin was found unexpectedly to be independent of cytosol translocation. Moreover, unlike CyaA-OVA E5, its processing retains the characteristics of the noncytosolic pathway due to its TAP independence 42 . Fusion proteins between LF and HIV gp120 were found to be presented in vitro to specific CD8+ cells in a protective Ag-dependent way. Lactacystin inhibition demonstrated that this presentation pathway involves proteasome processing 45 . Although the TAP dependence and the sensitivity to BFA inhibition were not established, it was speculated that the recombinant toxin is delivered into the cytosolic pathway.
In contrast to the adenylate cyclase of B. anthracis (a toxin different from LF but internalized in a similar mechanism), the adenylate cyclase of B. pertussis, CyaA, is internalized by a mechanism that does not require vacuolar acidification 22, 23 . Our results concerning the resistance of the CyaA-OVA E5 presentation to chloroquine are consistent with these earlier observations. This result excludes a putative endolysosomal degradation of CyaA-OVA E5 as a source of SIINFEKL peptide.
Compared with other toxins, the originality of CyaA stems from its translocation mechanism, apparently independent of receptor-mediated endocytosis. The translocation of CyaA occurs in two steps 16, 21, 25 . First, the C-terminal domain of CyaA binds to the external surface of plasma membrane with a mechanism requiring the posttranslational palmitoylation of Lys983 46 and possibly through interaction with gangliosides 22 . Second, the catalytic domain is translocated across the plasma membrane in a Ca2+-dependent manner 25 . We show, in the present study, that the presentation of CyaA-OVA E5 is also dependent on Ca2+ concentration. This last point establishes that delivery of CyaA-OVA E5 in the cytosolic pathway for MHC I presentation depends on the same mechanism of membrane translocation as the delivery of the catalytic domain of wild-type CyaA in target cells.
The strong immunogenicity of CyaA-LCMV E5 in vivo 29, 30 suggests
that this molecule could be presented by professional APC and
especially by dendritic cells that are thought to be the main APC
population involved in the priming of naive T cells in vivo 47 .
Indeed, we found that GM-CSF-induced BMDC and the FSDC line are able to
present efficiently CyaA-OVA E5 to B3Z CD8+ T cells. The
phenotypic heterogeneity of macrophage populations does not seem to
limit the presentation of CyaA-OVA E5 to B3Z, since both peritoneal
macrophages recruited by thioglycollate-induced inflammation of the
peritoneal cavity (Thio-PEC) and macrophages derived from bone marrow
precursors by culture in the presence of M-CSF are able to present
CyaA-OVA E5 to B3Z T cells. Moreover, unfractionated splenocytes are
also able to present CyaA-OVA E5 with a comparable efficiency. The
LB27.4 B cell lymphoma and the EL4 thymoma cell lines, as well as the
LKb fibroblastic cell line (Fig. 7 and data not shown),
also present CyaA-OVA E5 but with a reduced efficiency for reasons that
remain to be clarified. Thus, taken together, our results demonstrate
that the presentation of CyaA-OVA E5 to CD8+ T cells is not
restricted to professional APC. If the variety of cell types
intoxicable in vitro by wild-type CyaA 16 is well established, little
is known, to our knowledge, on the cell types targeted by CyaA in vivo
during the B. pertussis infection or after an immunization
with the recombinant CyaA toxin. Experiments are currently being
performed to clarify the role of the different cell types (from
lymphoid and myeloid origin) involved in the presentation of detoxified
recombinant CyaA both in vitro and in vivo. However, the lack of
cellular specificity distinguishes unequivocally the CyaA presentation
pathway from the previously described pathways for the presentation of
exogenous Ags and is consistent with the supposed absence of cellular
receptor for this toxin.
In the mouse model, CyaA has been described to be a major virulence factor during the initial phases of B. pertussis infection 48, 49 . Moreover, it was demonstrated that immunization with purified CyaA can protect mice from B. pertussis infection 50, 51 . Our results strongly suggest that intoxication of cells with wild-type CyaA toxin could result in the activation of MHC class I-restricted CD8+ T cells specific for the toxin itself. Mills et al. 52 have shown that the transfer of CD8+ T cells from immune mice to immunosuppressed naive recipient increases the bacterial load in a respiratory model of infection, whereas CD4+ T cells confer protection. Thus, the exact role of CD8+ T cells in the B. pertussis infection remains to be clarified.
In conclusion, our results show that the presentation of recombinant CyaA to CD8+ T cells constitutes a new and unconventional access of an exogenous Ag to the cytosolic pathway. These findings are relevant to the rational design of recombinant CD8+ T cell vaccines based on the invasivity of the CyaA molecule and to the understanding of the immune response raised against CyaA during the B. pertussis infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claude Leclerc, Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; CyaA, adenylate cyclase toxin of Bordetella pertussis; CyaA-OVA E5, genetically detoxified CyaA carrying the OVA CTL epitope; LCMV, lymphocytic choriomeningitis virus; CyaA-LCMV E5, genetically detoxified CyaA carrying the lymphocytic choriomeningitis virus CTL epitope; BMM, bone marrow-derived macrophage; BMDC, bone marrow-derived dendritic cell; Thio-PEC, thioglycollate-induced peritoneal exudate cells; CM, complete medium; M-CSF, macrophage CSF; GM-CSF, granulocyte-macrophage CSF; CCB, cytochalasine B; DMA, dimethyl amiloride; BFA, brefeldin A; LLnL, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal; LF, lethal factor.
Received for publication July 29, 1998. Accepted for publication October 29, 1998.
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C. Fayolle, A. Osickova, R. Osicka, T. Henry, M.-J. Rojas, M.-F. Saron, P. Sebo, and C. Leclerc Delivery of Multiple Epitopes by Recombinant Detoxified Adenylate Cyclase of Bordetella pertussis Induces Protective Antiviral Immunity J. Virol., August 15, 2001; 75(16): 7330 - 7338. [Abstract] [Full Text] [PDF]
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 P. Guermonprez, N. Khelef, E. Blouin, P. Rieu, P. Ricciardi-Castagnoli, N. Guiso, D. Ladant, and C. Leclerc The Adenylate Cyclase Toxin of Bordetella pertussis Binds to Target Cells via the {alpha}M{beta}2 Integrin (Cd11b/Cd18) J. Exp. Med., May 7, 2001; 193(9): 1035 - 1044. [Abstract] [Full Text] [PDF]
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N. Haicheur, E. Bismuth, S. Bosset, O. Adotevi, G. Warnier, V. Lacabanne, A. Regnault, C. Desaymard, S. Amigorena, P. Ricciardi-Castagnoli, et al. The B Subunit of Shiga Toxin Fused to a Tumor Antigen Elicits CTL and Targets Dendritic Cells to Allow MHC Class I-Restricted Presentation of Peptides Derived from Exogenous Antigens J. Immunol., September 15, 2000; 165(6): 3301 - 3308. [Abstract] [Full Text] [PDF]
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G. Dadaglio, Z. Moukrim, R. Lo-Man, V. Sheshko, P. Sebo, and C. Leclerc Induction of a Polarized Th1 Response by Insertion of Multiple Copies of a Viral T-Cell Epitope into Adenylate Cyclase of Bordetella pertussis Infect. Immun., July 1, 2000; 68(7): 3867 - 3872. [Abstract] [Full Text] [PDF]
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T. Basar, P. Guermonprez, M. Rojas, and C. Leclerc Delivery of CD8+ T-Cell Epitopes into Major Histocompatibility Complex Class I Antigen Presentation Pathway by Bordetella pertussis Adenylate Cyclase: Delineation of Cell Invasive Structures and Permissive Insertion Sites Infect. Immun., January 1, 2000; 68(1): 247 - 256. [Abstract] [Full Text] [PDF]
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 A. Osicková, E. Maier, and R. Benz An Amphipathic alpha -Helix Including Glutamates 509 and 516 Is Crucial for Membrane Translocation of Adenylate Cyclase Toxin and Modulates Formation and Cation Selectivity of Its Membrane Channels J. Biol. Chem., December 31, 1999; 274(53): 37644 - 37650. [Abstract] [Full Text] [PDF]
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