Identification of dendritic cell subsets responding to genital infection by Chlamydia muridarum

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum . Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naïve T cells in vivo . In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro . However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro . Our findings show that these two DC subsets likely have different functions in vivo . cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.     

Distinct functional capacities of mouse thymic and splenic dendritic cell populations

Dendritic cells (DC) are antigen-presenting cells that activate naive T cells. Murine DC are a heterogeneous population and can be subdivided into distinct subsets with different immune regulatory functions, namely the conventional DC (cDC), which include the CD8(+)Sirpalpha(-) and CD8(-)Sirpalpha(+) DC, and the plasmacytoid DC (pDC). In this study, the phenotype and function of DC subsets in both the thymus and spleen were compared. Significant differences between the thymic and splenic DC were observed in the expression of genes encoding chemokine receptors (CCRs), toll-like receptors (TLRs) and chemokines. Thymic DC expressed high levels of genes encoding a unique set of chemokines (CCL17 and CCL22) known to be important for T-cell development. Moreover, the capacity of the DC from the two organs to produce IL-6, IFN-alpha and IL-12p70 in response to the TLR 9 agonist CpG differed markedly, indicating intrinsic functional differences between subsets with similar surface phenotype. These results indicate that the microenvironment is an important factor that contributes to the functional specification of DC subsets in different lymphoid tissues.     

The Virion Host Shut-Off (vhs) Protein Blocks a TLR-Independent Pathway of Herpes Simplex Virus Type 1 Recognition in Human and Mouse Dendritic Cells

Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell''s susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.     

The role of type I interferon production by dendritic cells in host defense

Type I interferons (IFN) and dendritic cells (DC) share an overlapping history, with rapidly accumulating evidence for vital roles for both production of type 1 IFN by DC and the interaction of this IFN both with DC and components of the innate and adaptive immune responses. Within the innate immune response, the plasmacytoid DC (pDC) are the "professional" IFN producing cells, expressing specialized toll-like receptors (TLR7 and -9) and high constitutive expression of IRF-7 that allow them to respond to viruses with rapid and extremely robust IFN production; following activation and production of IFN, the pDC subsequently mature into antigen presenting cells that help to shape the adaptive immune response. However, like most cells in the body, the myeloid or conventional DC (mDC or cDC) also produce type I IFNs, albeit typically at a lower level than that observed with pDC, and this IFN is also important in innate and adaptive immunity induced by these classic antigen presenting cells. These two major DC subsets and their IFN products interact both with each other as well as with NK cells, monocytes, T helper cells, T cytotoxic cells, T regulatory cells and B cells to orchestrate the early immune response. This review discusses some of the converging history of DC and IFN as well as mechanisms for IFN induction in DC and the effects of this IFN on the developing immune response.     

Dendritic cells infiltrating human non-small cell lung cancer are blocked at immature stage

The efficacy of immune response to control human cancer remains controversial. It is particularly debated whether and to what extent the capacity of tumor-infiltrating dendritic cells (DC) to drive immunization can be turned off by transformed cells, leading to tumor-specific tolerance rather than immunization. To address this issue, we have characterized the DC isolated from human non-small cell lung cancer (NSCLC). These biopsy specimens contained CD11c(high) myeloid DC (mDC), but also CD11c(-) plasmacytoid DC (pDC) and a third DC subset expressing intermediate level of CD11c. Compared with peripheral blood, CD11c(high) tumor-infiltrating DC (TIDC) displayed a "semi-mature" phenotype, and TLR4 or TLR8 stimulation drove them to mature partially and to secrete limited amounts of cytokines. In contrast, most tumor-infiltrating pDC were immature but underwent partial maturation after TLR7 activation, whereas TLR9 ligation triggered low secretion of IFN-alpha. CD11c(int) mDC represented approximately 25% of total DC in tumoral and peritumoral tissues and expressed low levels of costimulatory molecules contrasting with high levels of the immunoinhibitory molecule B7-H1. Finally, the poor APC function of total TIDC even after TLR stimulation and the migratory response of both tumor-infiltrating mDC and pDC toward CCL21 and SDF-1 in vitro suggested their ability to compromise the tumor-specific immune response in draining lymph nodes in vivo. Further studies will be required to establish the specific role of the three TIDC subsets in tumor immunity and to draw conclusions for the design of therapeutic strategies.     

Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei

Melioidosis sepsis, caused by Burkholderia pseudomallei, is associated with high mortality due to an overwhelming inflammatory response. Plasmacytoid dendritic cells (pDC) are potent producers of type I interferons (IFN). This study investigated whether pDC and type I IFN play a role during the early stages of B. pseudomallei infection. Human and murine pDC internalised and killed B. pseudomallei as efficiently as murine conventional DC (cDC). pDC derived from B. pseudomallei-susceptible (BALB/c) mice demonstrated poor intracellular killing and increased IFN-alpha compared to pDC derived from B. pseudomallei-resistant (C57BL/6) mice. This is the first evidence of pDC bactericidal activity against B. pseudomallei infection.     

Collaborative action of NF-kappaB and p38 MAPK is involved in CpG DNA-induced IFN-alpha and chemokine production in human plasmacytoid dendritic cells

CpG DNA induces plasmacytoid dendritic cells (pDC) to produce type I IFN and chemokines. However, it has not been fully elucidated how the TLR9 signaling pathway is linked to these gene expressions. We examined the mechanisms involving the TLR9 and type I IFN signaling pathways, in relation to CpG DNA-induced IFN-alpha, IFN regulatory factor (IRF)-7, and chemokines CXCL10 and CCL3 in human pDC. In pDC, NF-kappaB subunits p65 and p50 were constitutively activated. pDC also constitutively expressed IRF-7 and CCL3, and the gene expressions seemed to be regulated by NF-kappaB. CpG DNA enhanced the NF-kappaB p65/p50 activity, which collaborated with p38 MAPK to up-regulate the expressions of IRF-7, CXCL10, and CCL3 in a manner independent of type I IFN signaling. We then examined the pathway through which IFN-alpha is expressed. Type I IFN induced the expression of IRF-7, but not of IFN-alpha, in a NF-kappaB-independent way. CpG DNA enabled the type I IFN-treated pDC to express IFN-alpha in the presence of NF-kappaB/p38 MAPK inhibitor, and chloroquine abrogated this effect. With CpG DNA, IRF-7, both constitutively and newly expressed, moved to the nuclei independently of NF-kappaB/p38 MAPK. These findings suggest that, in CpG DNA-stimulated human pDC, the induction of IRF-7, CXCL10, and CCL3 is mediated by the NF-kappaB/p38 MAPK pathway, and that IRF-7 is activated upstream of the activation of NF-kappaB/p38 MAPK in chloroquine-sensitive regulatory machinery, thereby leading to the expression of IFN-alpha.     

Identification of Toll-Like Receptor 9 as Parapoxvirus Ovis-Sensing Receptor in Plasmacytoid Dendritic Cells

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and co-stimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.     

Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses

Optimal Ag targeting and activation of APCs, especially dendritic cells (DCs), are important in vaccine development. In this study, we report the effects of different Toll-like receptor (TLR)-binding compounds to enhance immune responses induced by human APCs, including CD123(+) plasmacytoid DCs (PDCs), CD11c(+) myeloid DCs (MDCs), monocytes, and B cells. PDCs, which express TLR7 and TLR9, responded to imidazoquinolines (imiquimod and R-848) and to CpG oligodeoxynucleotides stimulation, resulting in enhancement in expression of costimulatory molecules and induction of IFN-alpha and IL-12p70. In contrast, MDCs, which express TLR3, TLR4, and TLR7, responded to poly(I:C), LPS, and imidazoquinolines with phenotypic maturation and high production of IL-12 p70 without producing detectable IFN-alpha. Optimally TLR ligand-stimulated PDCs or MDCs exposed to CMV or HIV-1 Ags enhanced autologous CMV- and HIV-1-specific memory T cell responses as measured by effector cytokine production compared with TLR ligand-activated monocytes and B cells or unstimulated PDCs and MDCs. Together, these data show that targeting specific DC subsets using TLR ligands can enhance their ability to activate virus-specific T cells, providing information for the rational design of TLR ligands as adjuvants for vaccines or immune modulating therapy.     

TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcepsilonRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-alpha when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an approximately 15-fold reduction in FcepsilonRIalpha mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-alpha also caused a significant approximately 4-fold reduction in FcepsilonRIalpha mRNA. Finally, this reduction in FcepsilonRIalpha mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.     

Differential pattern recognition receptor expression but stereotyped responsiveness in rat spleen dendritic cell subsets

Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity. In the rat, spleen DC can be separated into three morphological and phenotypical distinct subsets, namely CD4+, CD4-, and plasmacytoid DC (pDC), whose frequencies are strain dependent. We correlated the expression of TLR and nucleotide-binding oligomerization domain 2 (NOD2) in these DC subsets to their in vitro responsiveness to specific ligands. CD4- DC expressed high levels of TLR1, 2, 3, and 10 mRNA, low TLR4, 5, 6, 7, and 9, and very low, if any, TLR8. pDC had a restricted repertoire characterized by high TLR7 and 9. CD4+ DC expressed all TLR and 10-fold higher levels of NOD2 mRNA than CD4- and pDC. Upon stimulation by TLR and NOD2 ligands, each DC subset responded in quite a stereotyped fashion. TLR2/6, 3, 4, 5, 9, and NOD2 triggering induced CD4- DC to mature and produce high IL-12p40, low IL-10, and TNF-alpha. TLR7/8 and 9 triggering induced pDC to mature and produce copious amounts of IL-6, IL-12p40, and TNF-alpha and low IFN-alpha. CD4+ DC were very poor producers of inflammatory cytokines. This study suggests that the nature of spleen DC responses to pathogens is dependent on subset specific-stimulation rather than intrinsic plasticity.     

Plasmacytoid DC from aged mice down-regulate CD8 T cell responses by inhibiting cDC maturation after Encephalitozoon cuniculi infection

Age associated impairment of immune function results in inefficient vaccination, tumor surveillance and increased severity of infections. Several alterations in adaptive immunity have been observed and recent studies report age related declines in innate immune responses to opportunistic pathogens including Encephalitozoon cuniculi. We previously demonstrated that conventional dendritic cells (cDC) from 9-month-old animals exhibit sub-optimal response to E. cuniculi infection, suggesting that age associated immune senescence begins earlier than expected. We focused this study on how age affects plasmacytoid DC (pDC) function. More specifically how aged pDC affect cDC function as we observed that the latter are the predominant activators of CD8 T cells during this infection. Our present study demonstrates that pDC from middle-aged mice (12 months) suppress young (8 week old) cDC driven CD8 T cell priming against E. cuniculi infection. The suppressive effect of pDC from older mice decreased maturation of young cDC via cell contact. Aged mouse pDC exhibited higher expression of PD-L1 and blockade of their interaction with cDC via this molecule restored cDC maturation and T cell priming. Furthermore, the PD-L1 dependent suppression of cDC T cell priming was restricted to effector function of antigen-specific CD8 T cells not their expansion. To the best of our knowledge, the data presented here is the first report highlighting a cell contact dependent, PD-L1 regulated, age associated defect in a DC subpopulation that results in a sub-optimal immune response against E. cuniculi infection. These results have broad implications for design of immunotherapeutic approaches to enhance immunity for aging populations.     

IRF family proteins and type I interferon induction in dendritic cells

Dendritic cells （DC）, although a minor population in hematopoietic cells, produce type I interferons （IFN） and other cytokines and are essential for innate immunity. They are also potent antigen presenters and regulate adaptive immunity. Among DC subtypes plasmacytoid DC （pDC） produce the highest amounts of type I IFN. In addition, pro- and anti-inflammatory cytokines such as IL-12 and IL-10 are induced in DC in response to Toll like receptor （TLR） signaling and upon viral infection. Proteins in the IRF family control many aspects of DC activity. IRF-8 and IRF-4 are essential for DC development. They differentially control the development of four DC subsets. IRF-8^-/- mice are largely devoid of pDC and CD8α^＋ DC, while IRF-4^-/- mice lack CD4^＋ DC. IRF-8^-/-, IRF4^-/-, double knock-out mice have only few CD8α CD4^-DC that lack MHC Ⅱ. IRF proteins also control type Ⅰ IFN induction in DC. IRF-7, activated upon TLR signaling is required for IFN induction not only in pDC, but also in conventional DC （cDC） and non-DC cell types. IRF-3, although contributes to IFN induction in fibroblasts, is dispensable in IFN induction in DC. Our recent evidence reveals that type Ⅰ IFN induction in DC is critically dependent on IRF-8, which acts in the feedback phase of IFN gene induction in DC. Type Ⅰ IFN induction in pDC is mediated by MyD88 dependent signaling pathway, and differs from pathways employed in other cells, which mostly rely on TLR3 and RIG-Ⅰ family proteins. Other pro-inflammatory cytokines are produced in an IRF-5 dependent manner. However, IRF-5 is not required for IFN induction, suggesting the presence of separate mechanisms for induction of type Ⅰ IFN and other pro-inflammatory cytokines. IFN and other cytokines produced by activated DC in turn advance DC maturation and change the phenotype and function of DC. These processes are also likely to be governed by IRF family proteins.     

Cutting edge: generation of splenic CD8+ and CD8- dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8(+) and CD8(-) conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RA(high) plasmacytoid DC, two distinct CD24(high) and CD11b(high) cDC subsets were present, and these subsets showed equivalent properties to splenic CD8(+) and CD8(-) cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-alpha; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-alpha, MIP-1alpha, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24(high) subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.     

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists,imiquimod and resiquimod

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.     

Major subsets of human dendritic cells are efficiently transduced by self-complementary adeno-associated virus vectors 1 and 2

Dendritic cells (DC) are antigen-presenting cells pivotal for inducing immunity or tolerance. Gene transfer into DC is an important strategy for developing immunotherapeutic approaches against infectious pathogens and cancers. One of the vectors previously described for the transduction of human monocytes or DC is the recombinant adeno-associated virus (rAAV), with a genome conventionally packaged as a single-stranded (ss) molecule. Nevertheless, its use is limited by the poor and variable transduction efficiency of DC. In this study, AAV type 1 (AAV1) and AAV2 vectors, which expressed the enhanced green fluorescent protein and were packaged as ss or self-complementary (sc) duplex strands, were used to transduce different DC subsets generated ex vivo and the immunophenotypes, states of differentiation, and functions of the subsets were carefully examined. We show here for the first time that a single exposure of monocytes (M(o)) or CD34(+) progenitors (CD34) to sc rAAV1 or sc rAAV2 leads to high transduction levels (5 to 59%) of differentiated M(o)-DC, M(o)-Langerhans cells (LC), CD34-LC, or CD34-plasmacytoid DC (pDC), with no impact on their phenotypes and functional maturation of these cells, compared to those of exposure to ss rAAV. Moreover, we show that all these DC subpopulations can also be efficiently transduced after commitment to their differentiation pathways. Furthermore, these DC subsets transduced with sc rAAV1 expressing a tumor antigen were potent activators of a CD8(+)-T-cell clone. Altogether, these results show the high potential of sc AAV1 and sc AAV2 vectors to transduce ex vivo conventional DC, LC, or pDC or to directly target them in vivo for the design of new DC-based immunotherapies.     

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands

We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002), and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC) were measured by evaluating CD86, CD40, and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV- individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-alpha and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-alpha. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV- individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.     

Plasmacytoid dendritic cells synergize with myeloid dendritic cells in the induction of antigen-specific antitumor immune responses

Plasmacytoid dendritic cells (pDC) are capable of producing high levels of type I IFNs upon viral stimulation, and play a central role in modulating innate and adaptive immunity against viral infections. Whereas many studies have assessed myeloid dendritic cells (mDC) in the induction of antitumor immune responses, the role of pDC in antitumor immunity has not been addressed. Moreover, the interaction of pDC with other dendritic cell subsets has not been evaluated. In this study, we analyzed the capacity of pDC in stimulating an Ag-specific T cell response. Immunization of mice with Ag-pulsed, activated pDC significantly augmented Ag-specific CD8(+) CTL responses, and protected mice from a subsequent tumor challenge. Immunization with a mixture of activated pDC plus mDC resulted in increased levels of Ag-specific CD8(+) T cells and an enhanced antitumor response compared with immunization with either dendritic cell subset alone. Synergy between pDC and mDC in their ability to activate T cells was dependent on MHC I expression by mDC, but not pDC, suggesting that pDC enhanced the ability of mDC to present Ag to T cells. Our results demonstrate that pDC and mDC can interact synergistically to induce an Ag-specific antitumor immune response in vivo.