Adenylate cyclase toxin translocates across target cell membrane without forming a pore

The adenylate cyclase toxin‐haemolysin of Bordetella (CyaA) targets CD11b⁺ myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation‐selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch‐clamp assays, we show that a combination of substitutions within the pore‐forming (E570Q) and acylation‐bearing domain (K860R) ablates selectively the cell‐permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC^‐ toxoid on CD11b‐expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8⁺ T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I‐restricted presentation and induce in vivo an OVA‐specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.     

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA−), or the BpCyaA− strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.     

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.     

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4⁺CD25⁺ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4⁺CD25⁺Foxp3⁺ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4⁺CD25⁺Foxp3⁺ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.     

cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.     

Bordetella Adenylate Cyclase Toxin Promotes Calcium Entry into Both CD11b+ and CD11b? Cells through cAMP-dependent L-type-like Calcium Channels

Adenylate cyclase toxin (ACT), a 200 kDa protein, is an essential virulence factor for Bordetella pertussis, the bacterium that causes whooping cough. ACT is a member of the pore-forming RTX (repeats-in-toxin) family of proteins that share a characteristic calcium-binding motif of Gly- and Asp-rich nonapeptide repeats and a marked cytolytic or cytotoxic activity. In addition, ACT exhibits a distinctive feature: it has an N-terminal calmodulin-dependent adenylate cyclase domain. Translocation of this domain into the host cytoplasm results in uncontrolled production of cAMP, and it has classically been assumed that this surge in cAMP is the basis for the toxin-mediated killing. Several members of the RTX family of toxins, including ACT, have been shown to induce intracellular calcium increases, through different mechanisms. We show here that ACT stimulates a raft-mediated calcium influx, through its cAMP production activity, that activates PKA, which in turn activates calcium channels with L-type properties. This process is shown to occur both in CD11b⁺ and CD11b⁻ cells, suggesting a common mechanism, independent of the toxin receptor. We also show that this ACT-induced calcium influx does not correlate with the toxin-induced cytotoxicity.     

Bordetella pertussis adenylate cyclase toxin modulates innate and adaptive immune responses: distinct roles for acylation and enzymatic activity in immunomodulation and cell death

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-alpha, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.     

Neutrophil-dendritic cell interaction plays an important role in live attenuated Leishmania vaccine induced immunity

BackgroundNeutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen^-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen^-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo.Methodology/findingsLdCen^-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen^-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen^-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4⁺T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen^-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen^-/- parasites by DCs. Neutrophil depletion in LdCen^-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4⁺Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4⁺ T cells in vivo.ConclusionsCollectively, LdCen^-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.     

Calcium,Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.     

CD103+CD11b+ Dendritic Cells Induce Th17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.     

Bordetella adenylate cyclase toxin: a swift saboteur of host defense

Bordetella that infect mammals produce a multifunctional repeat in toxin (RTX) adenylate cyclase toxin known as CyaA, an excellent example of bacterial sophistication in subverting host defense. Recent reports show that interaction of CyaA with tracheal epithelial cells aids adhesion of Bordetella to ciliated mucosa and induces production of the pro-inflammatory cytokine interleukin, IL-6. Myeloid phagocytes, attracted to the site of infection are the target of freshly secreted CyaA that binds to the alpha(M)beta2 integrin (CD11b/CD18), penetrates cells and promptly suppresses their bactericidal functions by converting cellular ATP to cAMP. Such uncontrolled cAMP signaling can also drive CD11b-expressing immature dendritic cells into a semi-mature state, possibly hijacking them to shape the local adaptive immune response towards tolerance of the pathogen.     

Mycobacterium paratuberculosis CobT Activates Dendritic Cells via Engagement of Toll-like Receptor 4 Resulting in Th1 Cell Expansion

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4⁺/CD8⁺CD44^highCD62L^low memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.     

Aging impacts CD103+CD8+ T cell presence and induction by dendritic cells in the genital tract

As women age, susceptibility to systemic and genital infections increases. Tissue‐resident memory T cells (TRMs) are CD103⁺CD8⁺ long‐lived lymphocytes that provide critical mucosal immune protection. Mucosal dendritic cells (DCs) are known to induce CD103 expression on CD8⁺ T cells. While CD103⁺CD8⁺ T cells are found throughout the female reproductive tract (FRT), the extent to which aging impacts their presence and induction by DCs remains unknown. Using hysterectomy tissues, we found that endometrial CD103⁺CD8⁺ T cells were increased in postmenopausal compared to premenopausal women. Endometrial DCs from postmenopausal women were significantly more effective at inducing CD103 expression on allogeneic naïve CD8⁺ T cells than DCs from premenopausal women; CD103 upregulation was mediated through membrane‐bound TGFβ signaling. In contrast, cervical CD103⁺ T cells and DC numbers declined in postmenopausal women with age. Decreases in DCs correlated with decreased CD103⁺ T cells in endocervix, but not ectocervix. Our findings demonstrate a previously unrecognized compartmentalization of TRMs in the FRT of postmenopausal women, with loss of TRMs and DCs in the cervix with aging, and increased TRMs and DC induction capacity in the endometrium. These findings are relevant to understanding immune protection in the FRT and to the design of vaccines for women of all ages.     

A novel in vivo inducible dendritic cell ablation model in mice

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c⁺ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c⁺ cells (iDTRΔ mice). We successfully deleted CD11c⁺ cells in bone marrow-derived DCs in vitro and splenic CD11c⁺ cells in vivo after DT treatment in iDTRΔ mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.     

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I–V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca²⁺-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132–1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562–1681). Despite deletion of 267 internal residues of the RTX domain, the Ca²⁺-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ_1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295–1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.     

CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8⁺ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8⁺ T cells recognize the epitope YopE_69-77. The features of the interaction between pathogen and host that result in this large CD8⁺ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE_69-77-specific CD8⁺ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE_69-77-specific CD8⁺ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α⁺ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE_69-77-specific CD8⁺ T cell expansion because this response was significantly reduced in Ccr2^-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE_69-77-specific CD8⁺ T cells ex vivo and promoted the expansion of YopE_69-77-specific CD8⁺ T cells in infected Ccr2^-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8⁺ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE_69-77-specific CD8⁺ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.