Optimizing DC Vaccination by Combination With Oncolytic Adenovirus Coexpressing IL-12 and GM-CSF

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CC-chemokine ligand 21 (CCL21⁺) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4⁺CD25⁺ T-cells and vascular endothelial growth factor (VEGF) expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-ΔB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.     

Therapeutic Immunity by Adoptive Tumor-primed CD4+ T-cell Transfer in Combination With In Vivo GITR Ligation

Tumor-primed CD4⁺ T cells from splenocytes of tumor-rejection mice in combination with in vivo glucocorticoid-induced tumor necrosis factor receptor (GITR) ligation (the combination therapy) elicited effective host CD8⁺ T cell–dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4⁺ T-cell activity and partially abrogated regulatory T cells (Treg) suppressor function. Dendritic cells (DCs) from tumor-draining lymph nodes (TDLNs) of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced interleukin (IL)-12 ex vivo. Whereas tumor-primed CD4⁺ T cells or in vivo GITR ligation alone induced a tumor-specific interferon (IFN)-γ-producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Treg''s ability to suppress IL-12 production by DCs and IFN-γ production by effectors ex vivo. Importantly, tumor-primed CD4⁺ CD25⁻ T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T-cell responses, and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.     

Cross-presentation of glycolipid from tumor cells loaded with alpha-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells

We report a mechanism to induce combined and long-lived CD4⁺ and CD8⁺ T cell immunity to several mouse tumors. Surprisingly, the initial source of antigen is a single low dose of tumor cells loaded with α-galactosylceramide (α-GalCer) glycolipid (tumor/Gal) but lacking co-stimulatory molecules. After tumor/Gal injection intravenously (i.v.), innate NKT and NK cells reject the tumor cells, some of which are taken up by dendritic cells (DCs). The DCs in turn cross-present glycolipid on CD1d molecules to NKT cells and undergo maturation. For B16 melanoma cells loaded with α-GalCer (B16/Gal), interferon γ–producing CD8⁺ T cells develop toward several melanoma peptides, again after a single low i.v. dose of B16/Gal. In all four poorly immunogenic tumors tested, a single dose of tumor/Gal i.v. allows mice to become resistant to tumors given subcutaneously. Resistance requires CD4⁺ and CD8⁺ cells, as well as DCs, and persists for 6–12 mo. Therefore, several immunogenic features of DCs are engaged by the CD1d-mediated cross-presentation of glycolipid-loaded tumor cells, leading to particularly strong and long-lived adaptive immunity.     

Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses

The induction of persistent intraepithelial CD8⁺ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8⁺ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8⁺ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8⁺ T cells were intra- or subepithelial, expressed α_E-integrin CD103, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a sphingosine-1-phosphate analog (FTY720), we found that the primed CD8⁺ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers 1,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8⁺ T cells but failed to induce intraepithelial CD103⁺CD8⁺ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8⁺ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8⁺ T cells.     

Harnessing Oncolytic Virus-mediated Antitumor Immunity in an Infected Cell Vaccine

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte–monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSV''s extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.     

Selective Recruitment of Immature and Mature Dendritic Cells by Distinct Chemokines Expressed in Different Anatomic Sites

DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34⁺ hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony–stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-α or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34⁺ HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3α, but also to MIP-1α and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-α, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3β. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3α and MIP-3β, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3β responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3β is also acquired by CD11c⁺ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3α mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3β mRNA specifically in T cell–rich areas, suggests a role for MIP-3α/CCR6 in recruitment of immature DCs at site of injury and for MIP-3β/CCR7 in accumulation of antigen-loaded mature DCs in T cell–rich areas.     

Mycobacterium tuberculosis infection induces il12rb1 splicing to generate a novel IL-12Rβ1 isoform that enhances DC migration

RNA splicing is an increasingly recognized regulator of immunity. Here, we demonstrate that after Mycobacterium tuberculosis infection (mRNA) il12rb1 is spliced by dendritic cells (DCs) to form an alternative (mRNA) il12rb1Δtm that encodes the protein IL-12Rβ1ΔTM. Compared with IL-12Rβ1, IL-12Rβ1ΔTM contains an altered C-terminal sequence and lacks a transmembrane domain. Expression of IL-12Rβ1ΔTM occurs in CD11c⁺ cells in the lungs during M. tuberculosis infection. Selective reconstitution of il12rb1^−/− DCs with (mRNA) il12rb1 and/or (mRNA) il12rb1Δtm demonstrates that IL-12Rβ1ΔTM augments IL-12Rβ1-dependent DC migration and activation of M. tuberculosis-specific T cells. It cannot mediate these activities independently of IL12Rβ1. We hypothesize that M. tuberculosis-exposed DCs express IL-12Rβ1ΔTM to enhance IL-12Rβ1-dependent migration and promote M. tuberculosis–specific T cell activation. IL-12Rβ1ΔTM thus represents a novel positive-regulator of IL12Rβ1-dependent DC function and of the immune response to M. tuberculosis.The control of M. tuberculosis infection occurs through an acquired antigen-specific CD4⁺ T cell response and the IL12B gene is essential to this response (Cooper et al., 2007; Cooper, 2009). Although IL-12 plays an expected role in modulating Th1 responses to M. tuberculosis, we have also shown that IL-12(p40)₂ is required for DCs to migrate in response to chemokines after exposure to mycobacterial and other pathogenic stimuli (Khader et al., 2006; Robinson et al., 2008). That this migration may be important in initiating T cell responses is suggested by the observation that depletion of CD11c⁺ cells before infection delays T cell activation and influences the outcome of infection (Tian et al., 2005).IL-12 family members mediate their biological activities through specific, high-affinity dimeric receptors. All these receptors share IL-12Rβ1, a 100-kD glycosylated protein that spans the plasma membrane and serves as a low-affinity receptor for the IL-12p40 subunit of IL-12 family members (Chua et al., 1994, 1995); coexpression with IL-12Rβ2 or IL-23R results in high-affinity binding of IL-12 and IL-23, respectively, and confers biological responsiveness to these cytokines (Presky et al., 1996; van Rietschoten et al., 2000; Parham et al., 2002). Polymorphisms in IL12B or IL12RB1 are associated with psoriasis (Capon et al., 2007), atopic dermatitis, and other allergic phenotypes (Takahashi et al., 2005) and, importantly, nonfunctional IL12RB1 alleles predispose to mycobacterial susceptibility (Altare et al., 1998; de Jong et al., 1998; Filipe-Santos et al., 2006; Fortin et al., 2007). Thus, understanding how IL-12Rβ1 expression and IL-12Rβ1–dependent signaling is regulated has important implications for tuberculosis and may impact other diseases.Given that CD11c⁺ cells contribute to the control of M. tuberculosis infection (Tian et al., 2005) and that IL-12(p40)₂ is required for their migration in response to pathogenic stimuli (Khader et al., 2006; McCormick et al., 2008; Robinson et al., 2008), we sought to determine if IL-12Rβ1 is required for DC migration after exposure to this organism. In the course of these investigations we not only confirmed this hypothesis but also discovered that DCs express both IL-12Rβ1 and a novel IL-12Rβ1 splice variant (IL-12Rβ1ΔTM) in response to M. tuberculosis. This splice variant can be seen at the mRNA level in CD11c⁺ cells from the lungs of M. tuberculosis–infected mice and as a protein in the membrane of DCs. Importantly, we have determined that IL-12Rβ1ΔTM functions to enhance IL-12Rβ1–dependent DC migration and promote CD4⁺ T cell activation. This finding not only impacts our understanding of DC migration and IL-12Rβ1–dependent mycobacterial immunity it also reveals a previously unknown positive regulator of IL-12Rβ1–dependent events.     

Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

A functionally distinct subset of CD103⁺ dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3⁺ T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and α4β7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103⁺ DCs. CD103⁺ SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103⁺ DCs do not derive from a CD103⁻ SI-LP DC intermediate. The majority of CD103⁺ MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8⁺ and CD4⁺ T cells. In contrast, most CD103⁻ MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103⁺ DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103⁺ MLN DCs isolated from SB Crohn's patients. Thus, small intestinal CD103⁺ DCs represent a potential novel target for regulating human intestinal inflammatory responses.     

mRNA-transfected Dendritic Cells Expressing Polypeptides That Link MHC-I Presentation to Constitutive TLR4 Activation Confer Tumor Immunity

Recently, we have developed a novel genetic platform for improving dendritic cell (DC) induction of peptide-specific CD8 T cells, based on membrane-anchored β₂-microglobulin (β₂m) linked to a selected antigenic peptide at its N-terminus and to the cytosolic domain of toll-like receptor (TLR)4 C-terminally. In vitro transcribed mRNA transfection of antigen presenting cells resulted in polypeptides that efficiently coupled peptide presentation to cellular activation. In the present study, we evaluated the immunogenicity of such constructs in mRNA-transfected immature murine bone marrow-derived DCs. We show that the encoded peptide β₂m-TLR4 products were expressed at the cell surface up to 72 hours and stimulated the maturation of DCs. In vivo, these DCs prompted efficient peptide-specific T-cell activation and target cell killing which were superior to those induced by peptide-loaded, LPS-stimulated DCs. This superiority was also evident in the ability to protect mice from tumor progression following the administration of B16F10.9 melanoma cells and to inhibit the development of pre-established B16F10.9 tumors. Our results provide evidence that the products of two recombinant genes encoding for peptide-hβ₂m-TLR4 and peptide-hβ₂m-K^b expressed from exogenous mRNA can cooperate to couple Major Histocompatibility Complex (MHC-I) peptide presentation to TLR-mediated signaling, offering a safe, economical and highly versatile genetic platform for a novel category of CTL-inducing vaccines.     

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient''s human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8⁺ and CD4⁺ T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8⁺ response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4⁺ response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8⁺ and CD4⁺ T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.     

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141⁺ DC subset. CD141⁺ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c⁺ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141⁺ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8⁺ cytotoxic T lymphocytes than poly I:C–activated CD1c⁺ DCs. Importantly, CD141⁺ DCs, but not CD1c⁺ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141⁺ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α⁺ DC subset. The data demonstrate a role for CD141⁺ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.The essential role of DCs in the induction and regulation of immune responses to pathogens, self-antigens (Ags), and cancers is now well established. All DCs excel at processing and presenting Ag and priming naive T cell responses, but the complexity of DC subsets and their individual specialized functions is just becoming apparent (MacDonald et al., 2002; Villadangos and Schnorrer, 2007; Naik, 2008). Promising DC-based therapeutic vaccines have been described to treat malignancies and infections (Vulink et al., 2008), but the majority of these use in vitro–generated monocyte-derived DC (MoDC), and the physiological standing of this DC subtype is currently unclear. Understanding the emerging complexities of human DC subset biology is therefore essential to develop new vaccines and therapeutics targeting DC.The characterization and function of human DC subsets has been confounded by their rarity, the lack of distinctive markers, and limited access to human tissues. Human blood DCs comprise ∼1% of circulating PBMCs and have been classically defined as Ag-presenting leukocytes that lack other leukocyte lineage markers (CD3, 14, 15, 19, 20, and 56) and express high levels of MHC class II (HLA-DR) molecules (Hart, 1997). These can be broadly categorized into two groups: plasmacytoid CD11c⁻CD123⁺ DC and conventional or myeloid CD11c⁺CD123⁻ DC. We have described three further phenotypically distinct subsets of CD11c⁺ DC, defined by their expression of CD16, CD1c (BDCA-1), and CD141 (BDCA-3; MacDonald et al., 2002). Gene expression profiling and hierarchical clustering data has indicated that plasmacytoid DC and CD16⁺ DC arise from separate precursor cells, whereas the CD1c⁺ DC and CD141⁺ DC subsets appear to have a common origin and represent two different stages of a similar subset (Lindstedt et al., 2005). However, CD1c⁺ and CD141⁺ DCs each have unique gene expression profiles distinct from monocytes and MoDC, and this predicts that they have different functions (Dzionek et al., 2000; MacDonald et al., 2002; Lindstedt et al., 2005).The concept of distinct DC subtypes with unique capabilities to influence immunological outcomes is exemplified by the mouse CD8α⁻ and CD8α⁺ conventional DC subsets that reside in the lymph nodes and spleen (Villadangos and Schnorrer, 2007; Naik, 2008). The CD8α⁻ DC subset appears to be most effective at inducing Th2 responses (Maldonado-López et al., 1999; Pulendran et al., 1999) and processing and presenting Ag to CD4⁺ T cells via the MHC class II pathway (Pooley et al., 2001; Dudziak et al., 2007; Villadangos and Schnorrer, 2007). In contrast, the CD8α⁺ DC subset has a unique ability to take up dead or dying cells and to process and present exogenous Ag on MHC class I molecules to CD8⁺ T cells (i.e., cross-presentation; den Haan et al., 2000; Iyoda et al., 2002; Schnorrer et al., 2006). There is now substantial evidence that the CD8α⁺ DC subset plays a crucial role in the induction of protective CD8⁺ CTL responses that are essential for the eradication of cancers, viruses, and other pathogenic infections (Dudziak et al., 2007; Hildner et al., 2008; López-Bravo and Ardavín, 2008; Naik, 2008). The identification of the human DC subset with similar functional capacity would be a significant advance and would enable translation of mouse DC biology into clinical practice.Correlation of the human and mouse DC subsets has been hampered by differences in their defining markers (human DCs do not express CD8α). Interestingly, computational genome-wide expression profiling clustered human CD141⁺ DC and CD1c⁺ DC with the mouse CD8α⁺ and CD8α⁻ conventional DC subsets, respectively (Robbins et al., 2008). Human CD141⁺ DC and mouse CD8α⁺ DC share a number of phenotypic similarities, including expression of Toll-like receptor (TLR) 3 (Edwards et al., 2003; Lindstedt et al., 2005), the novel surface molecule Necl2 (nectin-like protein 2; Galibert et al., 2005), and the C-type lectin CLEC9A (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). Thus, whether the human CD141⁺ DC subset is the human functional equivalent of the mouse CD8α⁺ DC subset has now become a major question for immunologists.CD141⁺ DCs constitute only ∼0.03% of human PBMCs and, although present in other human tissues, their low proportions and difficulties with aseptic human tissue access mean that they have never been isolated in sufficient quantity to study their function until now. We report the first detailed functional analysis of human CD141⁺ DCs in response to TLR3 stimuli and define their role in the induction of Th1 responses and cross-presentation.     

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

Human BDCA3⁺ dendritic cells (DCs) were suggested to be homologous to mouse CD8α⁺ DCs. We demonstrate that human BDCA3⁺ DCs are more efficient than their BDCA1⁺ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8⁺ T cells, which is similar to mouse CD8α⁺ DCs as compared with CD11b⁺ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α⁺, human BDCA3⁺, and sheep CD26⁺ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8⁺ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8⁺ T lymphocytes. Finally, XCR1^−/− mice have decreased early CD8⁺ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α⁺ DCs in higher vertebrates and promotes their ability to activate early CD8⁺ T cell defenses against an intracellular pathogenic bacteria.DCs are central to immune defenses in mammals. In mice, three subsets of DCs are resident of lymphoid organs (Crozat et al., 2010). Plasmacytoid DCs (pDCs) are professional producers of IFN-α and -β, contributing to immune defenses against viruses (Baranek et al., 2009). CD11b⁺ DCs preferentially prime CD4⁺ T cells and promote humoral immunity (Carter et al., 2006; Dudziak et al., 2007). CD8α⁺ DCs are endowed with a unique efficiency in priming CD8⁺ T cells and in cross-presenting exogenous antigens (Carter et al., 2006; Dudziak et al., 2007). CD8α⁺ DCs are required for the natural induction of strong CD8⁺ T cell responses against tumors (Hildner et al., 2008; Sancho et al., 2008) or West Nile virus (Hildner et al., 2008). Specific delivery of vaccine antigens to CD8α⁺ DCs is especially efficient for vaccination against intracellular pathogens or tumors (Bonifaz et al., 2004; Nchinda et al., 2008). Therefore, identification of human DC subsets functionally homologous to mouse CD8α⁺ DCs (CD8α⁺-type DCs) should be a major step forward for the design of innovative vaccination or immunotherapeutic strategies against cancer or infections (Crozat et al., 2010). So far, no conserved marker has been identified to specifically and unambiguously define CD8α⁺-type DCs in several mammalian species. Although human BDCA3⁺ and mouse CD8α⁺ DCs express the C-type lectin CLEC9A, which is known to be involved in cross-presentation in mice, this marker is also found on some human CD14⁺ monocytes and on mouse pDCs (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). It is of note that no orthologue of CLEC9A has been identified yet in non mammalian vertebrate species.We have recently performed comparative genomics studies of human, mouse, and sheep DC subsets to help identify potential homologies between these cell types across mammalian species (Robbins et al., 2008; unpublished data). We found that human blood BDCA3⁺ DCs share a specific gene signature with mouse CD8α⁺ DCs and proposed that they could be human professional cross-presenting DCs (Robbins et al., 2008; Crozat et al., 2010). In this paper, we demonstrate that human BDCA3⁺ DCs are more potent than their BDCA1⁺ counterparts or than pDCs for CD8⁺ T cell activation through antigen cross-presentation. We established elsewhere that sheep lymph CD26⁺ DCs (Epardaud et al., 2004) are also equivalents to mouse CD8α⁺ DCs based on gene expression and functions such as superior efficacy for presentation of soluble antigen to CD8⁺ T cells (unpublished data). In this paper, we identify the XC chemokine receptor 1 (XCR1) as the first universal marker specifically expressed by the CD8α⁺-type DCs from three different mammalian species: ovine CD26⁺ DCs, mouse CD8α⁺ DCs, and human BDCA3⁺ DCs. We show that the Xcr1 gene is present and well conserved in all higher vertebrates from reptiles to human. The ligand of XCR1, chemokine (C motif) ligand 1 (XCL1), is specifically expressed by activated NK and CD8⁺ T cells in mouse and human. We show that Xcl1 mRNA is stored selectively in memory CD8⁺ T cells, allowing them to rapidly produce high levels of this chemokine upon stimulation. Finally, we show that XCR1^−/− mice have decreased CD8⁺ T cell responses to Listeria monocytogenes (Lm) associated with higher bacterial loads early after infection. Overall, our study strongly suggests an important and conserved role in mammals for XCR1 in the cross talk between NK or CD8⁺ T cells and CD8α⁺-type DCs, identifying this molecule as a novel tool to survey and target the DCs endowed with the best cross-presentation capacity across species.     

Immature Dendritic Cells Phagocytose Apoptotic Cells via αvβ5 and CD36, and Cross-present Antigens to Cytotoxic T Lymphocytes

Dendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present viral, tumor, and self-antigens to CD8⁺ T cells. This in vitro pathway corresponds to the in vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and that this process is accompanied by the expression of a unique profile of receptors, in particular the α_vβ₅ integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than DCs, and although they express many receptors that mediate this uptake, they lack the α_vβ₅ integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major histocompatibility complex. We suggest that the α_vβ₅ integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway.     

In liver fibrosis,dendritic cells govern hepatic inflammation in mice via TNF-α

Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b⁺CD8^– fraction, a lower B220⁺ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-α. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease.     

HLA-DR+ leukocytes acquire CD1 antigens in embryonic and fetal human skin and contain functional antigen-presenting cells

Adequate numbers and functional maturity are needed for leukocytes to exhibit a protective role in host defense. During intrauterine life, the skin immune system has to acquire these prerequisites to protect the newborn from infection in the hostile external environment after birth. We investigated the quantitative, phenotypic, and functional development of skin leukocytes and analyzed the factors controlling their proliferation and trafficking during skin development. We show that CD45⁺ leukocytes are scattered in embryonic human skin and that their numbers continuously increase as the developing skin generates an environment that promotes proliferation of skin resident leukocytes as well as the influx of leukocytes from the circulation. We also found that CD45⁺HLA-DR^highCD1c⁺ dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor β1 production precedes Langerin and CD1a expression on CD45⁺CD1c⁺ Langerhans cell (LC) precursors. Functionally, embryonic antigen-presenting cells (APCs) are able to phagocytose antigen, to up-regulate costimulatory molecules upon culture, and to efficiently stimulate T cells in a mixed lymphocyte reaction. Collectively, our data provide insight into skin DC biology and the mechanisms through which skin DCs presumably populate the skin during development.     

Arming Cytokine-induced Killer Cells With Chimeric Antigen Receptors: CD28 Outperforms Combined CD28–OX40 “Super-stimulation”

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8⁺CD56⁺ cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of “younger” T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ–OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ–OX40 CAR accelerated terminal maturation of CD56⁺ CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56⁻ phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ–OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; “super-costimulation” by the CD28-ζ–OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.     

Indirect Recruitment of a CD40 Signaling Pathway in Dendritic Cells by B7-DC Cross-Linking Antibody Modulates T Cell Functions

The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40^−/− DCs treated with B7-DC XAb (DC^XAb) activated DAP12, but failed to activate NFκB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D₃. CD40^−/− DC^XAb also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DC^XAb to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant.     

Dendritic Cells Retrovirally Transduced with a Model Antigen Gene Are Therapeutically Effective against Established Pulmonary Metastases

Dendritic cells (DCs) are bone marrow–derived leukocytes that function as potent antigen presenting cells capable of initiating T cell–dependent responses from quiescent lymphocytes. DC pulsed with tumor-associated antigen (TAA) peptide or protein have recently been demonstrated to elicit antigen-specific protective antitumor immunity in a number of murine models. Transduction of DCs with TAA genes may allow stable, prolonged antigen expression as well as the potential for presentation of multiple, or unidentified, epitopes in association with major histocompatibility complex class I and/or class II molecules. To evaluate the potential efficacy of retrovirally transduced DCs, bone marrow cells harvested from BALB/c mice were transduced with either a model antigen gene encoding β-galactosidase (β-gal) or a control gene encoding rat HER-2/neu (Neu) by coculture with irradiated ecotropic retroviral producer lines. Bone marrow cells were differentiated into DC in vitro using granulocyte/macrophage colony-stimulating factor and interleukin-4. After 7 d in culture, cells were 45–78% double positive for DC phenotypic cell surface markers by FACS^® analysis, and DC transduced with β-gal were 41–72% positive for β-gal expression by X-gal staining. In addition, coculture of β-gal transduced DC with a β-gal–specific T cell line (CTLx) resulted in the production of large amounts of interferon-γ, demonstrating that transduced DCs could process and present endogenously expressed β-gal. DC transduced with β-gal and control rat HER-2/neu were then used to treat 3-d lung metastases in mice bearing an experimental murine tumor CT26.CL25, expressing the model antigen, β-gal. Treatment with β-gal–transduced DC significantly reduced the number of pulmonary metastatic nodules compared with treatment with Hank''s balanced salt solution or DCs transduced with rat HER-2/neu. In addition, immunization with β-gal–transduced DCs resulted in the generation of antigen-specific cytotoxic T lymphocytes (CTLs), which were significantly more reactive against relevant tumor targets than CTLs generated from mice immunized with DCs pulsed with the L^d-restricted β-gal peptide. The results observed in this rapidly lethal tumor model suggest that DCs transduced with TAA may be a useful treatment modality in tumor immunotherapy.Dendritic cells (DCs)¹ are highly specialized APCs that possess unique immunostimulatory properties and function as the principal activators of quiescent T cells, and thus cellular immune responses in vivo. (1). These bone marrow–derived leukocytes express a unique repertoire of cell-surface molecules including high levels of MHC class I and II, adhesion molecules, and costimulatory molecules, all of which assist in the activation of T cells. As motile cells with elaborate cytoplasmic processes and a unique veiled morphology, DCs are specialized for antigen capture and transport from the periphery to T cell–dependent areas of lymphoid organs.The key role of DCs in the initiation of immune responses has focused the attention of many investigators on the potential efficacy of these cells in tumor immunotherapy. Several groups have demonstrated that DCs pulsed with peptides from tumor associated antigens (TAA) can induce antigen-specific antitumor responses in vivo in a variety of murine tumor models (2–7). The successes of TAA-pulsed DCs in murine models has supported the use of autologous, peptide-pulsed DCs in recent clinical trials (8).In developing strategies to optimize the use of DCs in tumor immunotherapy, retroviral transduction of DCs with TAA genes may offer important advantages over peptide-pulsed DCs and other methods of immunization currently in use. The efficacy of peptide-pulsed DCs might be limited in vivo, because peptides pulsed onto DCs stay bound to the MHC molecules only transiently due to variation in peptide binding affinities, peptide–MHC complex dissociation, and MHC turnover (9). Additionally, the use of peptide-pulsed DCs is dependent on the knowledge of the HLA haplotype of the patient, as well as the restriction element of the peptide epitopes for any particular antigen.However, retroviral transduction of DCs with TAA genes may allow for constitutive expression of the full-length protein leading to prolonged antigen presentation in vivo, and presentation of multiple or unidentified antigen epitopes in the context of MHC class I, and possibly class II, molecules. Additionally, retrovirally transduced DCs are entirely autologous, thus abrogating the potential for development of neutralizing antibodies with repeated treatments, as can occur with recombinant viral immunization modalities. TAA-transduced DCs might also be given repeatedly and/or combined with other viral or peptide-based immunization strategies.As nonreplicating, terminally differentiated cells, mature DCs are poor candidates for retroviral gene modification. However, dividing bone marrow progenitor cells can be efficiently transduced with retroviral vectors (10–12). Because DCs can successfully be generated in vitro from bone marrow cells in the presence of GM-CSF–containing cytokine combinations (13–16), we used a method by which bone marrow cells were retrovirally transduced by coculture with irradiated producer lines and then differentiated in vitro to DCs. This method has previously been shown to be effective in human DC by retroviral transduction of CD34⁺ hematopoietic progenitor cells (HPCs) and differentiation of transduced cells in vitro to mature DC (17, 18).In this study, we demonstrate that murine DCs retrovirally transduced with the gene encoding β-galactosidase (β-gal) stably express, process, and present the gene in the context of MHC class I molecules, and that treatment with β-gal–transduced DCs is capable of mediating effective antitumor activity against established pulmonary metastases of a murine tumor expressing β-gal.