Conventional DCs Research Articles

Differential requirements of MHC and of DCs for endogenous proliferation of different T cell subsets in vivo (46.16)

Abstract T cells transferred into severe lymphopenic hosts undergo rapid proliferative responses that are distinct from conventional homeostatic proliferation, known as 'endogenous proliferation'. Unlike homeostatic proliferation, cytokines such as IL-7 is dispensable, yet TCR interaction with peptide:MHC complexes is believed to be essential for this process to occur. However, cell types inducing the proliferation have not been defined. In this study, we provide evidence that CD11c+ conventional DCs play pivotal roles in inducing endogenous proliferation of both naive and memory phenotype CD4 T cells via TCR-MHC II interaction. T cells transferred into MHC II-/- TCRβ-/- or CD11c+ DC-deleted TCRβ-/- recipients failed to undergo endogenous proliferation. By contrast, CD8 T cell endogenous proliferation was independent of MHC I or CD11c+ DCs. Interestingly, depleting both B cells and DCs was able to abrogate the proliferation of naive CD8 T cells in TCRβ-/- environments. However, memory phenotype CD8 T cell proliferation was not abrogated by the absence of both B cells and DCs. Taken together, these results strongly suggest that depending on the T cell lineages as well as the differentiation status different mechanisms operate during endogenous proliferation, revealing unexpected in vivo complexity of T cell proliferation under lymphopenic conditions.

CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation

CX3CR1 expression is associated with the commitment of CSF-1R+ myeloid precursors to the macrophage/dendritic cell (DC) lineage. However, the relationship of the CSF-1R+ CX3CR1+ macrophage/DC precursor (MDP) with other DC precursors and the role of CX3CR1 in macrophage and DC development remain unclear. We show that MDPs give rise to conventional DCs (cDCs), plasmacytoid DCs (PDCs), and monocytes, including Gr1+ inflammatory monocytes that differentiate into TipDCs during infection. CX3CR1 deficiency selectively impairs the recruitment of blood Gr1+ monocytes in the spleen after transfer and during acute Listeria monocytogenes infection but does not affect the development of monocytes, cDCs, and PDCs.

TLR4-Agonist Stimulated Human Multipotent Mesenchymal Stromal Cells Enhance Both Hematopoietic Progenitors and Myeloid Differentiation

Human mesenchymal stromal cells (MSCs) can replicate as undifferentiated cells in vitro and are capable of differentiating into multiple mesenchymal tissues. In the bone marrow, MSCs are thought to contribute to nurturing stroma involved in maintaining and modulating hematopoiesis. Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells, and their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like autoimmune disease. In inflammatory conditions like sepsis, cytokines like G-CSF are elevated in serum, CD34+ cells increase in circulation, and bone marrow myelopoiesis is enhanced. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs. In vitro cultured bone marrow MSCs were analyzed for mRNA expression of TLRs. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional and plasmacytoid blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with TLR-4 agonists, mRNA transcripts of flt3-ligand and thrombopoietin increased, and MSCs secreted high amounts of G-CSF, GM-CSF, and M-CSF, whereas stimulation with TLR-9 agonists did not lead to changes in cytokine levels measured in supernatants. TLR-4 stimulated MSC conditioned media enhanced myeloid colony formation from human CD34+ cells about 2.5× fold compared to non-stimulated MSC conditioned media. Furthermore, in co-culture assays of MSCs with CD34+ cells, TLR-4 stimulated MSCs were capable to retain 8 fold more CD34+ cells in divisions 0–3 and maintained overall 2 fold higher numbers of CD34+ cells. In secondary colony formation assays, CD34+ cells from TLR-4 stimulated MSC co-cultures produced about 2.5 fold more myeloid colonies, with some being CFU-GEMM. Moreover, hematopoietic cells taken from TLR-4 stimulated MSC co-cultures were able to engraft conditioned newborn Rag 2−/− gc−/− mice and differentiated into B, T, and myeloid cells, while CD34+ cells from co-cultures of non-stimulated MSCs did not engraft.These findings thus suggest a regulatory mechanism how, e.g. in gram-negative infection, inflammatory signals are translated via stromal cells into sustained early hematopoiesis and myeloid differentiation to efficiently meet the requests of an ongoing innate immune response.

HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase–dependent mechanism

Plasmacytoid DCs (pDCs) have been implicated as crucial cells in antiviral immune responses. On recognizing HIV, they become activated, secreting large amounts of IFN-alpha and inflammatory cytokines, thereby potentiating innate and adaptive antiviral immune responses. Here, we have shown that HIV-stimulated human pDCs can also induce the differentiation of naive CD4+ T cells into Tregs with suppressive function. This differentiation was independent of pDC production of IFN-alpha and primarily dependent on pDC expression of indoleamine 2,3-dioxygenase, which was induced through the TLR/MyD88 pathway, following binding of HIV to CD4 and triggering of TLR7 by HIV genomic RNA. Functionally, the Tregs induced by pDCs were shown to inhibit the maturation of bystander conventional DCs. This study therefore reveals what we believe to be a novel mechanism by which pDC may regulate and potentially limit anti-HIV immune responses.

Murine plasmacytoid dendritic cells express the inhibitory FcγRIIB as their sole FcγR and fail to mediate antigen presentation of immune complexes to T cells

Plasmacytoid DC's (pDCs) in their role as Type I IFN producers, are key regulators of the innate immune response, yet their role in the adaptive immune response remains unclear. The ability of murine pDCs to present antigen in immune complexes (ICs) to T cells was examined. Unlike conventional DCs, ovalbumin‐IC exposed murine pDCs failed both to upregulate costimulatory molecules and to activate OVA specific CD4+ and CD8+ T cells. The failure of murine pDCs to promote T cell activation through either the exogenous or cross‐presentation pathways occurred despite IC binding and internalization. ICs internalized by pDCs however were inefficiently delivered to an intracellular degradative compartment. This defect in the IC processing capability is likely the consequence of a lack of activating Fc Receptor expression (FcRI, III, IV) and the exclusive expression of the inhibitory receptor FcRIIB. This selective expression of FcRIIB by pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. Thus, pDCs are unable to effectively process ICs for antigen presentation, and may not be able to directly activate naive T cells in vivo.

Plasmacytoid dendritic cells induce NK cell–dependent, tumor antigen–specific T cell cross-priming and tumor regression in mice

A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-gamma production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8(+) T cells.

M-CSF: a novel plasmacytoid and conventional dendritic cell poietin

The critical importance of plasmacytoid dendritic cells (pDCs) in viral infection, autoimmunity, and tolerance has focused major attention on these cells that are rare in blood and immune organs of humans and mice. The recent development of an Flt-3 ligand (FL) culture system of bone marrow cells has led to the simple generation of large numbers of pDCs that resemble their in vivo steady-state counterparts. The FL system has allowed unforeseen insight into the biology of pDCs, and it is assumed that FL is the crucial growth factor for these cells. Surprisingly we have found that a cell type with high capacity for interferon-alpha (IFN-alpha) production in response to CpG-containing oligonucleotides, a feature of pDCs, develop within macrophage-colony-stimulating factor (M-CSF)-generated bone marrow cultures. Analysis of this phenomenon revealed that M-CSF is able to drive pDCs as well as conventional DCs (cDCs) from BM precursor cells in vitro. Furthermore, application of M-CSF to mice was able to drive pDCs and cDCs development in vivo. It is noteworthy that using mice deficient in FL indicated that the M-CSF-driven generation of pDCs and cDCs in vitro and in vivo was independent of endogenous FL.

TLR Agonist Induced Cytokine Production in Human Multipotent Mesenchymal Stromal Cells: A Potential Mechanism How Hematopoiesis Is Enhanced during Generalized Inflammatory Conditions.

Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells. Their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like tissue damage and cancer. Human multipotent mesenchymal stromal cells (MSCs) have been shown to differentiate into various mesenchymal tissues such as bone, cartilage, and fat, as well as marrow and lymphoid organ stroma cells. Human MSCs are able to maintain CD34+ cells to some extent in vitro. Furthermore, it has been demonstrated that upon intra bone marrow transplantation into adult immunodeficient mice MSC derived cells support human hematopoiesis in vivo. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs in inflammatory conditions. MSCs from human bone marrow, cord blood, and umbilical cord whartons jelly were cultured by plastic adherence in IMDM 20% FCS, 1–8M dexamethasone (only during first 3 weeks), expanded for 2 passages, and subsequently analyzed. MSCs expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1, while G-CSF was rarely detectable. Consistently, respective cytokines were measured in supernatants at the following, declining levels (pg/ml): IL-6 (10000–10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF, whereas GM-CSF was rarely detectable, G-CSF, FLT3-L, and TPO were not detectable by ELISA. MSCs were further analyzed for expression of TLRs by semiquantitative RT-PCR. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional (BDCA-1+, CD14−, CD19−) and plasmacytoid (BDCA-4+,CD14−,CD19−) blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with LPS (TLR-4 agonist), MSCs produced previously undetectable G-CSF and GM-CSF, and M-CSF levels increased about 4 fold compared to untreated MSCs, whereas stimulation with CpG motifs (TLR-9 agonists) did not lead to changes in cytokine release detected in supernatants. In co-culture experiments using MSCs as a feeder layer for cord blood CD34+ cells, MSCs supported hematopoiesis and the development of myeloid cells. Importantly, MSC preconditioning with LPS led to an 1,7 fold increase in total hematopoietic cell number, while preconditioning with CpG had no measurable effect. These results demonstrate that MSCs express certain TLRs, and are capable to respond to pathogen associated molecules with an increase in secretion of hematopoiesis-relevant cytokines, and thus reveal a potential mechanism how hematopoiesis is enhanced during generalized inflammatory conditions, supporting e.g. in case of gram-negative sepsis efficient innate immune responses.

Type I Interferon Production during Herpes Simplex Virus Infection Is Controlled by Cell-Type-Specific Viral Recognition through Toll-Like Receptor 9, the Mitochondrial Antiviral Signaling Protein Pathway, and Novel Recognition Systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-alpha/beta) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-alpha/beta production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.

Plasmacytoid dendritic cells initiate a complex chemokine and cytokine network and are a viable drug target in chronic HCV patients

Plasmacytoid dendritic cells (pDCs) are the professional type I interferon (IFN)-producing cells, and upon activation they traffic to lymph organs, where they bridge innate and adaptive immunity. Using multianalyte profiling (MAP), we have mapped the key chemokines and cytokines produced in response to pDC activation, taking into consideration the role of autocrine IFN, as well as paracrine effects on other innate cells (e.g., monocytes and conventional DCs). Interestingly, we identify four distinct cytokine/chemokine loops initiated by Toll-like receptor engagement. Finally, we applied this analytic approach to the study of pDC activity in chronic hepatitis C patients. Based on the activation state of pDCs in fresh blood, the lack of agonistic activity of infectious virions, the production of a broad array of cytokines/chemokines once stimulated, and the direct effects of pDCs on other PBMCs, we conclude that the pDCs from hepatitis C virus (HCV)-infected individuals are fully functional and are, indeed, a viable drug target. In sum, this study provides insight into the use of MAP technology for characterizing cytokine networks, and highlights how a rare cell type integrates the activation of other inflammatory cells. Furthermore, this work will help evaluate the therapeutic application of pDC agonists in diseases such as chronic HCV infection.

Organ-dependent in vivo priming of naive CD4+,but not CD8+,T cells by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) play a pivotal role as cytokine-secreting accessory cells in the antimicrobial immune defense. In contrast, the capacity of PDCs to act as antigen-presenting cells in naive T cell priming remains unclear. By studying T cell responses in mice that lack conventional DCs (cDCs), and by the use of a PDC-specific antigen-targeting strategy, we show that PDCs can initiate productive naive CD4+ T cell responses in lymph nodes, but not in the spleen. PDC-triggered CD4+ T cell responses differed from cDC-driven responses in that they were not associated with concomitant CD8+ T cell priming. Our results establish PDCs as a bona fide DC subset that initiates unique CD4+ Th cell–dominated primary immune responses.

High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer

Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14(+) monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.

Innate Immune Response to Adenoviral Vectors Is Mediated by both Toll-Like Receptor-Dependent and -Independent Pathways

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.

Ikaros is required for plasmacytoid dendritic cell differentiation

AbstractPlasmacytoid dendritic cells (pDCs) are specialized DCs that produce high levels of type I IFN upon viral infection. Despite their key immunoregulatory role, little is known about pDC ontogeny or how developmental events regulate their function. We show that mice expressing low levels of the transcription factor Ikaros (IkL/L) lack peripheral pDCs, but not other DC subsets. Loss of pDCs is associated with an inability to produce type I IFN after challenge with Toll-like receptor-7 and -9 ligands, or murine cytomegalovirus (MCMV) infection. In contrast, conventional DCs are present in normal numbers and exhibit normal responses in vivo after challenge with MCMV or inactivated toxoplasma antigen. Interestingly, IkL/L bone marrow (BM) cells contain a pDC population that appears blocked at the Ly-49Q– stage of differentiation and fails to terminally differentiate in response to Flt-3L, a cytokine required for pDC differentiation. This differentiation block is strictly dependent on a cell-intrinsic requirement for Ikaros in pDC-committed precursors. Global gene expression profiling of IkL/L BM pDCs reveals an up-regulation of genes not normally expressed, or expressed at low levels, in WT pDCs. These studies suggest that Ikaros controls pDC differentiation by silencing a large array of genes.

A role for IFNγ in differential superantigen stimulation of conventional versus plasmacytoid DCs

Superantigens (SAgs) are known to play a role in food poisoning, toxic shock syndrome and have been identified as a potential mediator of autoimmunity. Although much is known about the effects of SAgs on T cells, by comparison few studies have investigated how SAgs influence innate immune cells. In particular no study has examined how SAgs affect murine plasmacytoid dendritic cells (pDC). We report that in vivo administration of staphylococcal enterotoxin A (SEA) increased the number of pDCs in secondary lymphoid organs, and induced CD86 and CD40 expression. Similar to SEA activation of conventional DCs (cDCs), pDCs relied on T cells, but not on CD40. Nonetheless, pDCs strictly required IFNγ for upregulation of CD86 and CD40, but cDCs did not depend upon IFNγ for activation. Further, even though IFNγ deficient pDCs were not activated by SEA, they were still capable of producing wild-type levels of IFNα in response to CpG oligodeoxynucleotide (ODN). The source of IFNγ for pDC activation was not T cells, nor did pDCs themselves have to synthesize or bind IFNγ, but the presence of IFNγ was essential. After SEA stimulation, IFNγ deficient mice fail to induce expression of the pDC dependent chemokines CXCL9, and demonstrated a defect in recruitment of pDCs to marginal zones of lymphoid organs. Thus, SEA exerts its combined effect on pDC activation, recruitment and chemokine induction through the action of IFNγ. This fundamental dichotomy of the effects of SAgs on pDCs versus cDCs show how a non-PAMP from bacteria, can selectively and indirectly stimulate innate cell subpopulations much in the same way that differential TLR expression influences cells of the innate immune system.

Toll-Like Receptor-Independent Triggering of Dendritic Cell Maturation by Viruses

The ability of vaccination to empower natural immune defenses against viral infection is well illustrated by the worldwide eradication of smallpox and the partial elimination of polio after global vaccination efforts (74, 98). Unfortunately, these major successes have been infrequent, and diverse viruses continue to threaten our societies. The development of new vaccines, or the improvement of existing ones, is currently hindered by a limited knowledge of the intracellular mechanisms leading to the activation of antiviral adaptive immunity and of the viral elements responsible for its triggering upon infection. The use of the viral RNA analog poly(I:C), the discovery of the highly virus-responsive plasmacytoid dendritic cells (pDCs), and the identification of Toll-like receptors (TLRs) specific for viral elements have led to a greater understanding of the initiation of antiviral immunity. Nevertheless, there is little evidence to suggest that these elements are sufficient—or even necessary—for adaptive antiviral immune responses. A more comprehensive understanding of the mechanisms by which viruses trigger the development of adaptive immunity is therefore still needed. Recent work led to the identification of a novel pathway for the activation of conventional DCs (cDCs), the primary cells responsible for the generation of adaptive immunity. The triggering of this pathway leads to strong adaptive antiviral immunity in vivo even in the absence of TLR signaling. Here, we review the evidence for the identification and characterization of this pathway. Intracellular molecular candidates that may participate in this pathway are discussed, and a model for the triggering of DC maturation by viruses is presented.

Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions

Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fcgamma receptor (FcgammaR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-gamma most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory FcgammaRs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory FcgammaRs on human DCs to generate either antigen-specific immunity or tolerance.

Spatial Restriction of Toll-Like Receptor Signals

Signals that cause production of type-1 interferon (IFN-α/β) constitute an important part of the mammalian immune response to microbial infection. Why this response is generated by plasmacytoid dendritic cells (pDCs) but not by other cells--even conventional dendritic cells--has been unclear. Microbial products are detected by the receptor TLR9 (Toll-like receptor 9), which activates a transcription factor complex that contains the transcription factor IRF-7 (interferon regulatory factor-7) and the adaptor protein MyD88. Honda et al. monitored localization of fluorescently tagged oligodeoxynucleotide ligands of TLR9 and found that a ligand that strongly activated IFN production, CpG-A, was localized in endosomes in cultured primary mouse pDCs, whereas a less effective ligand, CpG-B, appeared to be localized to lysosomes. MyD88 and IRF-7 were also localized in endosomes in cells exposed to CpG-A. In conventional DCs (cDCs), both ligands were associated with lysosomes and failed to strongly stimulate IFN production. However, if CpG-A was modified by formation of a complex with the cationic lipid 1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP), which caused it to be localized in endosomes of cDCs, IFN production was enhanced. It is not known exactly how TLR-9-bound CpG ODNs are retained in endosomes and how this is coordinated with release of the Myd88-IRF-7 complex to the nucleus, where it enhances IFN gene transcription. The authors note that more detailed study of signaling from endosomal vesicles and their trafficking may help explain how distinct forms of unmethylated DNA can specifically alter gene expression by modulating spatial restriction of signaling components. K. Honda, Y. Ohba, H. Yanai, H. Negishi, T. Mizutani, A. Takaoka, C. Taya, T. Taniguchi, Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434 , 1035-1040 (2005). [PubMed]

IFN Regulatory Factor-4 and -8 Govern Dendritic Cell Subset Development and Their Functional Diversity

Dendritic cells (DCs) are bone marrow (BM)-derived APCs central to both innate and adaptive immunity. DCs are a heterogeneous cell population composed of multiple subsets with diverse functions. The mechanism governing the generation of multiple DC subsets is, however, poorly understood. In this study we investigated the roles of closely related transcription factors, IFN regulatory factor (IRF)-4 and IRF-8, in DC development by analyzing IRF-4(-/-), IRF-8(-/-), and IRF-4(-/-)IRF-8(-/-) (double-knockout) mice. We found that IRF-4 is required for the generation of CD4(+) DCs, whereas IRF-8 is, as reported previously, essential for CD8alpha(+) DCs. Both IRFs support the development of CD4(-)CD8alpha(-) DCs. IRF-8 and, to a lesser degree, IRF-4 contribute to plasmacytoid DC (PDC) development. Thus, the two IRFs together regulate the development of all conventional DCs as well as PDCs. Consistent with these findings, IRF-4, but not IRF-8, was expressed in CD4(+) DCs, whereas only IRF-8 was expressed in CD8alpha(+) DCs. CD4(-)CD8alpha(-) DCs and PDCs expressed both IRFs. We also demonstrate in vitro that GM-CSF-mediated DC differentiation depends on IRF-4, whereas Fms-like tyrosine kinase 3 ligand-mediated differentiation depends mainly on IRF-8. Gene transfer experiments with double-knockout BM cells showed that both IRFs have an overlapping activity and stimulate a common process of DC development. Nonetheless, each IRF also possesses a distinct activity to stimulate subset-specific gene expression, leading to the generation of functionally divergent DCs. Together, IRF-4 and IRF-8 serve as a backbone of the molecular program regulating DC subset development and their functional diversity.

Rapid Activation of Spleen Dendritic Cell Subsets following Lymphocytic Choriomeningitis Virus Infection of Mice: Analysis of the Involvement of Type 1 IFN

In this study, we report the dynamic changes in activation and functions that occur in spleen dendritic cell (sDC) subsets following infection of mice with a natural murine pathogen, lymphocytic choriomeningitis virus (LCMV). Within 24 h postinfection (pi), sDCs acquired the ability to stimulate naive LCMV-specific CD8+ T cells ex vivo. Conventional (CD11chigh CD8+ and CD4+) sDC subsets rapidly up-regulated expression of costimulatory molecules and began to produce proinflammatory cytokines. Their tendency to undergo apoptosis ex vivo simultaneously increased, and in vivo the number of conventional DCs in the spleen decreased markedly, dropping approximately 2-fold by day 3 pi. Conversely, the number of plasmacytoid (CD11clowB220+) DCs in the spleen increased, so that they constituted almost 40% of sDCs by day 3 pi. Type 1 IFN production was up-regulated in plasmacytoid DCs by 24 h pi. Analysis of DC activation and maturation in mice unable to respond to type 1 IFNs implicated these cytokines in driving infection-associated phenotypic activation of conventional DCs and their enhanced tendency to undergo apoptosis, but also indicated the existence of type 1 IFN-independent pathways for the functional maturation of DCs during LCMV infection.