CD11c-DTR Transgenic Mice Research Articles

Systemic administration of β-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer

ABSTRACTConverting an immunosuppressive melanoma microenvironment into one that favors the induction of antitumor immunity is indispensable for effective cancer immunotherapy. In the current study we demonstrate that oat-derived β-(1-3)-(1-4)-glucan of 200 kDa molecular size (BG34-200) previously shown to mediate direct interaction with macrophages could alter the immune signature within melanoma microenvironment. Systemic administration of BG34-200 resulted in reversion of tolerant melanoma microenvironment to an immunogenic one that allows M1-type activation of macrophages, the induction of pro-inflammatory cytokines/chemokines including IFN-γ, TNF-α, CXCL9, and CXCL10, and enhanced IRF1 and PD-L1 expression. In turn, BG34-200 induced a superior antitumor response against primary and lung metastatic B16F10 melanoma compared to untreated controls. The enhanced tumor destruction was accompanied with significantly increased tumor infiltration of CD4+ and CD8+ T cells as well as elevated IFN-γ in the tumor sites. Systemic administration of BG34-200 also provoked systemic activation of tumor draining lymph node T cells that recognize antigens naturally expressing in melanoma (gp100/PMEL). Mechanistic studies using CD11b-knockout (KO), CD11 c-DTR transgenic mice and nude mice revealed that macrophages, DCs, T cells and NK cells were all required for the BG34-200-induced therapeutic benefit. Studies using IFN-γ-KO transgenic mice showed that IFN-γ was essential for the BG34-200-elicited antitumor response. Beyond melanoma, the therapeutic efficacy of BG34-200 and its immune stimulating activity were demonstrated in a mouse model of osteosarcoma. Together, BG34-200 is highly effective in modulating antitumor immunity. Our data support the potential therapeutic use of this novel immune modulator in the treatment of metastatic melanoma.

Exacerbation of Japanese Encephalitis by CD11chi Dendritic Cell Ablation Is Associated with an Imbalance in Regulatory Foxp3+ and IL-17+CD4+ Th17 Cells and in Ly-6Chi and Ly-6Clo Monocytes.

Japanese encephalitis (JE) is neuroinflammation characterized by uncontrolled infiltration of peripheral leukocytes into the central nervous system (CNS). We previously demonstrated exacerbation of JE following CD11chi dendritic cell (DC) ablation in CD11c-DTR transgenic mice. Moreover, CD11chi DC ablation led to abnormal differentiation of CD11b+Ly-6Chi monocytes and enhanced permeability of the blood-brain barrier (BBB), resulting in promoting the progression of JE. Here, we examined changes in lymphoid and myeloid-derived leukocyte subpopulations associated with pro- and anti-inflammation during JE progression. The analyses of this study focused on regulatory CD4+Foxp3+ regulatory T cells (Tregs), IL-17+CD4+ Th17 cells, and CD11b+Ly-6Chi and Ly-6Clo monocytes. CD11chi DC ablation resulted in the accumulation of IL-17+CD4+ Th17 cells in the CNS, thereby leading to lower ratio of Tregs to Th17 cells. This result was corroborated by the higher expression levels of IL-17 and RORγT in CD4+ T cells from the brains of CD11chi DC-ablated mice. In addition, CD11chi DC-ablated mice showed higher frequency and total number of inflammatory CD11b+Ly-6Chi monocytes, whereas CD11b+Ly-6Clo monocytes were detected with lower frequency and total number in CD11chi DC-ablated mice. Furthermore, CD11chi DC ablation altered the phenotype and function of CD11b+Ly-6Clo monocytes, resulting in lower levels of activation marker and anti-inflammatory cytokine (IL-10 and TGF-β) expression. Collectively, these results indicate that CD11chi DC ablation caused an imbalance in CD4+ Th17/Treg cells and CD11b+Ly-6Chi/Ly-6Clo monocytes in the lymphoid tissue and CNS during JE progression. This imbalanced orchestration of pro- and anti-inflammatory leukocytes following CD11chi DC ablation may contribute to the exacerbation of JE.

The Role of M2 Macrophages in the Progression of Chronic Kidney Disease following Acute Kidney Injury.

IntroductionAcute kidney injury (AKI) is a major risk factor in the development of chronic kidney disease (CKD). However, the mechanisms linking AKI to CKD remain unclear. We examined the alteration of macrophage phenotypes during an extended recovery period following ischemia/reperfusion injury (IRI) and determine their roles in the development of fibrosis.MethodsThe left renal pedicle of mice was clamped for 40 min. To deplete monocyte/macrophage, liposome clodronate was injected or CD11b-DTR and CD11c-DTR transgenic mice were used.ResultsThroughout the phase of IRI recovery, M2-phenotype macrophages made up the predominant macrophage subset. On day 28, renal fibrosis was clearly shown with increased type IV collagen and TGF-β. The depletion of macrophages induced by the liposome clodronate injection improved renal fibrosis with a reduction of kidney IL-6, type IV collagen, and TGF-β levels. Additionally, the adoptive transfer of the M2c macrophages partially reversed the beneficial effect of macrophage depletion, whereas the adoptive transfer of the M1 macrophages did not. M2 macrophages isolated from the kidneys during the recovery phase expressed 2.5 fold higher levels of TGF-β than the M1 macrophages. The injection of the diphtheria toxin into CD11b or CD11c-DTR transgenic mice resulted in lesser depletion or no change in M2 macrophages and had little impact on renal fibrosis.ConclusionAlthough M2 macrophages are known to be indispensible for short-term recovery, they are thought to be main culprit in the development of renal fibrosis following IRI.

Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors.

In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c+ dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.

OP0298 The Role of CD11c+ Dendritic Cells in Inflammatory Arthritis

BackgroundDendritic cells (DCs) play an important role in bridging the innate and the adaptive immune response by serving as antigen presenting cells and are therefore implicated in the initiation of...

Ovarian Dendritic Cells Act as a Double-Edged Pro-Ovulatory and Anti-Inflammatory Sword

Ovulation and inflammation share common attributes, including immune cell invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and fluorescence-activated cell sorting analyses, we demonstrate that CD11c-positive, F4/80-negative cells, representing DCs, are recruited to the ovary under gonadotropin regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of the cumulus-oocyte complex, release of the ovum from the ovarian follicle, formation of a functional corpus luteum, and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DC transplantation after conditional ablation of CD11c-positive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by up-regulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. In addition to discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classic immunologic role, which is relevant also to other biological systems.

Abstract 274: Lipid Accumulation in CD11c-Expressing Intimal Myeloid Cells Induces Chemokine Production Required for Leukocyte Recruitment to Early Atherosclerotic Lesions

Background: In the early stages atherosclerosis myeloid cells accumulate through the recruitment of circulating monocytes and macrophage proliferation. However, the first foam cells appear in the artery wall prior to increased monocyte recruitment and macrophage proliferation. These initial foam cells are derived from myeloid cells residing in the normal intima, and express integrin alpha X (CD11c). Rationale: When CD11c + cells were depleted at the initiation of a cholesterol-rich diet (CRD) in Ldlr -/- mice, lipid accumulation was reduced. We hypothesize that lipid accumulation in CD11c + intimal myeloid cells play an instrumental role in the recruitment of circulating monocytes to the artery wall. Methods: To assess the role if CD11c-expressing intimal myeloid cells in early atherosclerosis, CD11c + cells were deleted in Ldlr -/- mice reconstituted with bone marrow from CD11c-DTR transgenic mice. Mice reconstituted with C57BL/6 bone marrow were used as controls. Depletion of CD11c + cells was maintained throughout a 10 or 21 days CRD by giving injections of diphtheria toxin every three days. Monocyte recruitment was assayed by pulse labeling and quantifying BrdU positive leukocytes in the aortic arch. Intimal gene expression of adhesion molecules and chemokines was carried out by real-time PCR. Functional characterization of intimal chemokine expression on the recruitment of circulating monocytes was evaluated by antibody blockade of chemokine receptor CCR5 as well as reconstituting Ldlr -/- with CCL5 deficient bone marrow. Results: Recruitment of circulating monocytes was reduced in Ldlr-/- chimeras depleted of CD11c + cells. Correspondingly, intimal expression of chemokines CCL3, CCL4 and CCL5 were significantly blunted after 21 days of CRD in in Ldlr -/- BM chimeras depleted of CD11c + cells. The recruitment of BrdU labeled circulating monocytes was reduced by antibody blockade of CCR5 - the receptor for CCL3, CCL4 and CCL5. Conclusion: Collectively these data suggest that lipid accumulation in resident CD11c + intimal myeloid cells promote the recruitment of circulating monocytes to nascent lesions through the expression of CCL3, CCL4 and CCL5.

A8.3 The role of CD11C + dendritic cells in inflammatory arthritis

BackroundDendritic cells (DCs) play an important role in bridging the innate and the adaptive immune response by serving as antigen presenting cells and are therefore implicated in the initiation of...

AB0081 Cd11c+ dendritic cells play an important proinflammatory role in inflammatory arthritis

BackgroundDendritic cells (DCs) play an important role in bridging the innate and the adaptive immune response by serving as antigen presenting cells and are therefore implicated in the initiation of...

A Multifunctional Chimeric Chaperone Serves as a Novel Immune Modulator Inducing Therapeutic Antitumor Immunity

Converting the immunosuppressive tumor environment into one that is favorable to the induction of antitumor immunity is indispensable for effective cancer immunotherapy. Here, we strategically incorporate a pathogen (i.e., flagellin)-derived, NF-κB-stimulating "danger" signal into the large stress protein or chaperone Grp170 (HYOU1/ORP150) that was previously shown to facilitate antigen crosspresentation. This engineered chimeric molecule (i.e., Flagrp170) is capable of transporting tumor antigens and concurrently inducing functional activation of dendritic cells (DC). Intratumoral administration of adenoviruses expressing Flagrp170 induces a superior antitumor response against B16 melanoma and its distant lung metastasis compared with unmodified Grp170 and flagellin. The enhanced tumor destruction is accompanied with significantly increased tumor infiltration by CD8(+) cells as well as elevation of IFN-γ and interleukin (IL)-12 levels in the tumor sites. In situ Ad.Flagrp170 therapy provokes systemic activation of CTLs that recognize several antigens naturally expressing in melanoma (e.g., gp100/PMEL and TRP2/DCT). The mechanistic studies using CD11c-DTR transgenic mice and Batf3-deficient mice reveal that CD8α(+) DCs are required for the improved T-cell crosspriming. Antibody neutralization assays show that IL-12 and IFN-γ are essential for the Flagrp170-elicited antitumor response, which also involves CD8(+) T cells and natural killer cells. The therapeutic efficacy of Flagrp170 and its immunostimulating activity are also confirmed in mouse prostate cancer and colon carcinoma. Together, targeting the tumor microenvironment with this chimeric chaperone is highly effective in mobilizing or restoring antitumor immunity, supporting the potential therapeutic use of this novel immunomodulator in the treatment of metastatic diseases.

A Novel Pathway for CD8+ T Cell Activation by Donor Derived Non-Haematopoietic Cells Leading to Acute Allograft Rejection

Introduction: Although seminal studies have shown that CD8+ T cells can reject cardiac allografts that lack donor haematopoietic cells (HPCs) through a direct cognate interaction with graft parenchymal cells, the observation from other studies that secondary lymphoid tissue (SLT) is essential for graft rejection creates a paradox yet to be resolved. Here we examine the hypothesis that CD8+ T cell activation by graft parenchymal cells requires acquisition and re-presentation of intact MHC class I alloantigen by recipient antigen presenting cells (APCs) within SLT. If proven, this hypothesis would not only resolve this paradox, but would also represent the first definitive demonstration of the contribution of the semi-direct pathway to graft rejection. Methods: CD8+ T cell-mediated allograft rejection following activation by donor parenchymal cells was examined using a transgenic heterotopic murine heart transplant model. Balb/c cardiac donors were lethally irradiated and treated with depleting antibodies ensuring complete eradication of HPCs, such that parenchymal cells in these grafts were the only source of alloantigen presentation. These HPC-depleted Balb/c (Balb/cHPC-) cardiac allografts were transplanted into: 1) 2C transgenic mice (monoclonal population of CD8+ T cells against Ld MHC class I); 2) Splenectomised aly/aly (aly/alyspl) mice (complete absence of SLT) with adoptive transfer of 2C CD8+ T cells, to investigate the importance of SLT in this pathway; 3) CD11c-DTR transgenic mice, in which host dendritic cells (DCs) are selectively depleted by diphtheria toxin, allowing delineation of the role of these cells in parenchymal alloantigen presentation to CD8+ T cells. Results: 2C transgenic mice rejected Balb/cHPC- grafts as rapidly as non-depleted Balb/c grafts (MST = 5d vs. 4d respectively; p=0.4) suggesting an effective mechanism for parenchymal cell driven CD8+ T cell mediated rejection. Balb/cHPC- allografts showed prolonged survival (>50d) in aly/alyspl mice given 2C CD8+ T cells, whereas in non-splenectomised controls all grafts rejected (MST = 17d; p=0.01) suggesting an essential role for SLT in this pathway. In addition, when aly/alyspl mice were given activated 2C CD8+ T cells (from a 2C recipient of a Balb/c cardiac graft) they rapidly rejected Balb/cHPC- allografts (MST = 7d; p=0.01) indicating that SLT plays a fundamental role in the activation of CD8+ T cells. To examine, therefore, whether recipient DCs were required to present to CD8+ T cells within SLT, CD4+-depleted CD11c-DTR mice given 2C CD8+ T cells were transplanted with Balb/cHPC- allografts. When these mice were also treated with diphtheria toxin to deplete host DCs they demonstrated significantly delayed rejection kinetics when compared with untreated controls (MST = 26d vs. 16d; p=0.02) highlighting a key role for these cells. Conclusion: These results indicate that allorecognition of graft parenchymal cells represents an important mechanism of CD8+ T cell mediated rejection. They support the role of a novel pathway in which recipient DCs acquire intact MHC class I from donor parenchymal cells and present to CD8+ T cells in SLT leading to their activation.

Dendritic Cells Are Central Coordinators of the Host Immune Response to Staphylococcus aureus Bloodstream Infection

Dendritic cells (DCs) play an important role in integration of the immune responses induced by pathogens. The purpose of this study was to determine the importance of DCs in host defense against Staphylococcus aureus bacteremia. Using a murine infection model, we demonstrated that DCs are rapidly recruited into infected tissue after intravenous inoculation with S. aureus. The recruited DCs were fully functional and in a more advanced stage of maturation than those isolated from uninfected mice. Depletion of DCs in CD11c-DTR transgenic mice resulted in substantial worsening of infection, as indicated by increased bacterial loads in kidneys and lungs, accelerated mortality, and more severe pathology. Furthermore, DC depletion completely abolished IL-12 production in response to infection. The beneficial effect afforded by DCs during S. aureus infection was not mediated by their contribution to direct bacterial killing, nor by increased neutrophil recruitment. Instead, neutrophil influx (along with expression of CXC chemokines) was significantly enhanced in infected tissue after depletion of DCs. We also found that the bactericidal capacity of the recruited neutrophils was significantly impaired in DC-depleted mice. More importantly, the detrimental effect of DC depletion was practically reversed by treatment with exogenous recombinant mouse IL-12. Our results demonstrated that DCs, probably through their production of IL-12, play an important role in coordinating the inflammatory response during S. aureus infection.

Depletion of dendritic cells enhances susceptibility to cell-free but not cell-associated HTLV-1 infection in CD11c-DTR transgenic mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). The genesis of these diseases is believed to be associated with the route (mucosa versus blood), mode (cell-free versus cell-associated) of primary infection and eventual modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-DTR transgenic mouse model system that permits the conditional transient depletion of murine CD11c+ DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free, but not cell-associated HTLV-1 in both the CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA. Moreover, the depletion of DCs was found to significantly dampen the cellular immune response (IFN-γ+CD8+ T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free compared with late cell-associated HTLV-1 infection and highlight a significant aspect of viral immunopathogenesis related to the progression of ATL and HAM/TSP after the initial infection.

Murine Flt3L-derived dendritic cell mediated early immune responses are critical to controlling cell-free HTLV-1 infection

Human T-cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia. We observed previously that depletion of dendritic cells (DCs) in CD11c-DTR transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished the cellular immune response compared to mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host pathogen interaction between dendritic cells and cell-free HTLV-1, we used Fms-like tyrosine kinase 3 ligand (Flt3L) cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers (CD80, CD86, and MHC class II) on infection; however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3+ T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of interferon-stimulated genes, most cytokines, and transcription factors but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.

The tug-of-war between dendritic cells and human chronic viruses (105.47)

Abstract The human immune system is under constant challenge from many viruses, some of which the body is successfully able to clear. Other viruses have evolved to escape the host immune responses and thus persist, leading to the development of chronic diseases. Dendritic cells (DCs) are professional antigen presenting cells that play a major role in both innate and adaptive immunity against different pathogens. For the past few years our efforts have been focused on exploring the participation of DCs in the pathogenesis of HTLV-1, HIV-1 and HCV. We observed previously that depletion of DCs in CD11c-DTR transgenic mice enhanced the susceptibility to cell-free HTLV-1 infection. We further performed the host-pathogen interaction studies utilizing Flt3 ligand derived murine bone marrow DCs (FL-DCs). First, the kinetics of viral entry, proviral integration, and expression of the viral protein Tax was established and then effects of cell-free HTLV-1 was examined on these cells. Phenotypically, FL-DCs demonstrated activation and produced an array of proinflammatory cytokines as well as IFN-α. Virus-matured FL-DCs also stimulated proliferation of autologous CD3+ T cells and IFN-γ production. Gene expression studies revealed upregulation of interferon-stimulated genes, most cytokines, and transcription factors but a distinct downregulation of many chemokines. Overall, these results highlight the critical interaction of DCs with a human chronic virus important for the early immune responses.

Depletion of dendritic cells enhances susceptibility to cell-free infection of HTLV-1 in CD11c-DTR transgenic mice (45.11)

Abstract Human T-cell leukemia virus type 1 (HTLV-1) is associated with HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia. The genesis of these diseases is believed to be associated with the route and mode (cell-free versus cell-associated) of primary infection. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Mo-MLV to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-DTR transgenic mouse that permits conditional transient depletion of CD11c+ DCs. We infected these mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free, but not cell-associated HTLV-1 in both the CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA. Moreover, depletion of DCs significantly dampened the cellular immune response against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of ATL and HAM/TSP after the initial infection.

CCR6-Mediated Dendritic Cell Activation of Pathogen-Specific T Cells in Peyer's Patches

T cell activation by dendritic cells (DCs) is critical to the initiation of adaptive immune responses and protection against pathogens. Here, we demonstrate that a specialized DC subset in Peyer's patches (PPs) mediates the rapid activation of pathogen specific T cells. This DC subset is characterized by the expression of the chemokine receptor CCR6 and is found only in PPs. CCR6(+) DCs were recruited into the dome regions of PPs upon invasion of the follicle associated epithelium (FAE) by an enteric pathogen and were responsible for the rapid local activation of pathogen-specific T cells. CCR6-deficient DCs were unable to respond to bacterial invasion of PPs and failed to initiate T cell activation, resulting in reduced defense against oral infection. Thus, CCR6-dependent regulation of DCs is responsible for localized T cell dependent defense against entero-invasive pathogens.

Stimulation of CD8+T Cells following Diphtheria Toxin-Mediated Antigen Delivery into Dendritic Cells

Recognition and clearance of many intracellular pathogens requires the activation and subsequent effector functions of CD8+ T lymphocytes. To stimulate CD8+ T cells by immunization, the target antigens must be delivered into the cytosol of host cells. There they can be processed into peptides and presented in the context of major histocompatibility complex class I molecules to antigen-specific CD8+ T cells. One method of delivering antigens into the cytosol is to fuse them to modified bacterial toxins that are able to enter mammalian cells. The expression pattern of the toxin receptors in the host will determine the cell population that the toxin fusion protein targets and will thus restrict antigen-specific T-cell recognition to the same population. In this study we describe the development and characterization of a diphtheria toxin (DT)-based antigen delivery system. Using CD11c-DTR transgenic mice that express the DT receptor in dendritic cells (DC), this system allows for targeted delivery of CD8+ T-cell antigen to DC. We show that antigen-specific CD8+ T cells proliferate in CD11c-DTR mice following immunization with catalytically inactive DT-antigen fusion proteins. We also show that a toxin-based system that restricts antigen delivery to DC results in more robust antigen-specific CD8+ T-cell proliferation than a toxin-based system that does not restrict delivery to a particular cell type. These results have implications for vaccine design, and they suggest that use of a toxin-based vector to target antigen to DC may be an effective way to induce a CD8+ T-cell response.