Respiratory syncytial virus differentially activates murine myeloid and plasmacytoid dendritic cells

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis in young children. Upon infection both T helper 1 (Th1) and Th2 cytokines are produced. Because RSV-induced Th2 responses have been associated with severe immunopathology and aggravation of allergic reactions, the regulation of the immune response following RSV infection is crucial. In this study we examined the influence of RSV on the activation and function of murine bone marrow-derived dendritic cells (DCs). RSV induced the expression of maturation markers on myeloid DCs (mDCs) in vitro. The mDCs stimulated with RSV and ovalbumin (OVA) enhanced proliferation of OVA-specific T cells, which produced both Th1 and Th2 cytokines. In contrast to mDCs, RSV did not induce the expression of maturation markers on plasmacytoid DCs (pDCs), not did it enhance the proliferation of OVA-specific T cells that were cocultured with pDCs. However, RSV stimulated the production of interferon-alpha (IFN-alpha) by pDCs. Our findings indicate a clear difference in the functional activation of DC subsets. RSV-stimulated mDCs may have immunostimulatory effects on both Th1 and Th2 responses, while RSV-stimulated pDCs have direct antiviral activity through the release of IFN-alpha.     

Development of dendritic cell system

The dendritic cell system contains conventional dendritic cells (DCs) and plasmacytoid pre-dendritic cells (pDCs). Both DCs and pDCs are bone marrow derived cells. Although the common functions of DCs are antigen-processing and T-lymphocyte activation, they differ in surface markers, migratory patterns, and cytokine output. These differences can determine the fate of the T cells they activate. Several subsets of mature DCs have been described in both mouse and human and the developmental processes of these specialized DC subsets have been studied extensively. The original concept that all DCs were of myeloid origin was questioned by several recent studies, which demonstrated that in addition to the DCs derived from myeloid precursors, some DCs could also be efficiently generated from lymphoid-restricted precursors. Moreover, it has been shown recently that both conventional DCs and pDCs can be generated by the Flt3 expressing hemopoietic progenitors regardless of their myeloid- or lymphoid-origin. These findings suggest an early developmental flexibility of precursors for DCs and pDCs. This review summarizes some recent observations on the development of DC system in both human and mouse.     

Application of six-color flow cytometry for the assessment of dendritic cell responses in whole blood assays

Analysis of peripheral blood dendritic cells (PBDCs) is increasingly reaching clinical relevance in a wide range of pathologies, in which investigating the capacity of DC subsets to respond adequately to specific stimuli may aid the comprehension of underlying immunopathologic mechanisms. The evaluation of PBDC responses directly challenged in whole blood (WB) samples offers many advantages over other methods that require DC isolation and culture, but it is limited in multiparametric analysis, currently based on 3- or 4-color assays. Therefore, in this study we developed a 6-color assay dedicated to the analysis of PBDC responses upon WB stimulation. We incubated WB samples with ligands to toll-like receptors (TLRs) with a clear-cut distribution on myeloid DCs (mDCs) or plasmacytoid (pDCs) and analyzed DC responses in terms of upregulation of activation/maturation markers, as well as production of a wide range of regulatory cytokines. Four colors were used to gate on mDCs and pDCs that were identified as lineage-/HLA-DR+/CD11c+ and lineage-/HLA-DR+/CD123+, respectively, and two further colors were used to analyze either the surface expression of CD80, CD86, CD40 or CD83, or the intracellular accumulation of IL-12, tumor-necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IL-6, IL-10 or IL-4. With this method, we could directly compare in the same flow cytometric tube the responses of mDCs and pDCs to TLR stimulation, and investigate the reciprocal coexpression of distinct activation markers or regulatory cytokines. We suggest that the 6-color WB assay presented here may represent a novel tool for investigating the complex biology of DCs.     

Human Dendritic Cell Interactions with Whole Recombinant Yeast: Implications for HIV-1 Vaccine Development

Defects in number and function of dendritic cells (DCs) have been observed during HIV-1 infection, so therapeutic HIV-1 vaccine approaches that target or activate DCs may improve vaccine immunogenicity. To determine the potential of recombinant Saccharomyces cerevisiae yeast as an HIV-1 vaccine, we investigated interactions between yeast and human DCs. Yeast induced direct phenotypic maturation of monocyte-derived DCs (MDDCs) and enriched blood myeloid DCs (mDCs), but only indirectly matured blood plasmacytoid DCs (pDCs). Yeast-pulsed MDDCs and blood mDCs produced inflammatory cytokines and stimulated strong allo-reactive T cell proliferation. Both blood DC subsets internalized yeast, and when pulsed with yeast recombinant for HIV-1 Gag protein, both stimulated in vitro expansion of Gag-specific CD8⁺ memory T cells. These results suggest that S. cerevisiae yeast have potent adjuvant effects on human DCs. Furthermore, recombinant yeast-derived antigens are processed by human blood DCs for MHC class-I cross-presentation. These DC-targeting characteristics of yeast suggest that it may be an effective vaccine vector for induction of HIV-1-specific cellular immune responses.     

Quantitative analysis of chemokine expression by dendritic cell subsets in vitro and in vivo

Upon maturation, dendritic cells (DCs) have to adjust their chemokine expression to sequentially attract different leukocyte subsets. We used real-time quantitative polymerase chain reaction analysis to study in detail the expression of 12 chemokines involved in the recruitment of leukocytes into and inside secondary lymphoid organs, by DCs in distinct differentiation stages, both in vitro and in vivo. Monocyte-derived immature DCs expressed high levels of DC chemokine 1 (DC-CK1), EBI1-ligand chemokine (ELC), macrophage-derived chemokine (MDC), macrophage-inflammatory protein (MIP)-1alpha, and thymus and activation-regulated chemokine (TARC). Upon maturation, DCs up-regulated the expression of DC-CK1 (60-fold), ELC (7-fold), and TARC (10-fold). Activation of DCs by CD40 ligand further up-regulated the expression of ELC (25-fold). We found that freshly isolated blood DCs expressed only low levels of interleukin-8, lymphotactin, and MIP-1alpha. It is interesting that the chemokine profile expressed by activated CD11c(-) lymphoid-like as well as CD11c(+) myeloid blood DCs mimics that of monocyte-derived DCS: Additionally, purified Langerhans cells that had migrated out of the epidermis expressed a similar chemokine pattern. These data indicate that different DC subsets in vitro and in vivo can express the same chemokines to attract leukocytes.     

Chinese human immunodeficiency virus-1 patients with different routes of transmission exhibit altered expression levels of blood dendritic cell subpopulations

Dendritic cells (DCs) play a pivotal role in the pathogenesis of human immunodeficiency virus-1 (HIV-1). Reduced numbers of blood DCs have been observed in individuals with chronic HIV-1 infection. In the present study, we analyzed the expression levels of monocytes, myeloid dendritic cell (mDC) precursors, mDCs, and plasmacytoid dendritic cells (pDCs), in HIV-1-infected patients in China who were infected via different routes of transmission, including heterosexual and homosexual sexual contact, and blood transmission through importation of blood or blood products, to further elucidate their role in HIV. Compared with HIV-negative individuals (n?=?40), relative levels of CD11c+CD14?mDCs, CD11c++CD123(low) mDCs, and CD11c?CD123+ pDCs in total peripheral blood mononuclear cells (PBMCs) were significantly lower in all HIV patients (n?=?93), and in those with blood transmission (n?=?26) and heterosexual transmission (n?=?43), while relative levels of CD11c+CD14?mDCs were significantly lower in HIV patients infected via homosexual transmission (n?=?24). The results of correlation analysis demonstrated a significant negative correlation between CD4+ T-cell counts and the relative levels of CD11c++CD123(low) mDCs in HIV-I patients infected via blood transmission. There was no significant correlation between CD4+ T-cell counts and the expression level of other DC subpopulations in PBMCs from HIV patients. The results of this study suggest that HIV-1 patients with different routes of transmission exhibit altered expression levels of blood DC subpopulations, which contributes to dysregulated immune responses and pathogenesis of HIV-1.     

Human dendritic cell subsets display distinct interactions with the pathogenic mould Aspergillus fumigatus

The mould Aspergillus fumigatus is primarily an opportunistic pathogen of immunocompromised patients. Once fungal spores have been inhaled they encounter cells of the innate immune system, which include dendritic cells (DCs). DCs are the key antigen-presenting cells of the immune system and distinct subtypes, which differ in terms of origin, morphology and function.This study has systematically compared the interactions between A. fumigatus and myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and monocyte-derived DCs (moDCs). Analyses were performed by time-lapse video microscopy, scanning electron microscopy, plating assays, flow cytometry, 25-plex ELISA and transwell assays.The three subsets of DCs displayed distinct responses to the fungus with mDCs and moDCs showing the greatest similarities. mDCs and moDCs both produced rough convolutions and occasionally phagocytic cups upon exposure to A. fumigatus whereas pDCs maintained a smooth appearance. Both mDCs and moDCs phagocytosed conidia and germ tubes, while pDCs did not phagocytose any fungi. Analysis of cytokine release and maturation markers revealed specific differences in pro- and anti-inflammatory patterns between the different DC subsets.These distinct characteristics between the DC subsets highlight their differences and suggest specific roles of moDCs, mDCs and pDCs during their interaction with A. fumigatus in vivo.     

Regulation of the lifespan in dendritic cell subsets

The lifespan of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DCs in stimulating lymphocytes. Significant differences in the lifespan have been reported for various DC subsets, however, the molecular mechanisms for regulating such differences between DC subsets remain unclear. In this study, we compared the apoptosis signaling molecules in two major DC subjects, the myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). We observed a lower ratio between anti-apoptotic Bcl-2/Bcl-xL and pro-apoptotic Bax/Bak in shorter-lived myeloid DCs (mDCs) than in longer-lived plasmacytoid DCs (pDCs) or T cells. Transfection with Bcl-2 or Bcl-xL prolonged the survival of mouse primary mDCs in vitro, while deletion of Bcl-2 accelerated DC turnover in vivo. In addition, the ratios between anti-apoptotic Bcl-2/Bcl-xL and pro-apoptotic Bax/Bak could be regulated in DCs. Signaling from toll-like receptors (TLRs) up-regulated Bcl-xL and improved DC survival. Our data suggest that differential expression of apoptosis signaling molecules regulates the lifespan of different DC subsets.     

Dendritic cell dysregulation during HIV-1 infection

Dendritic cells (DCs) are a diverse subset of innate immune cells that are key regulators of the host response to human immunodeficiency virus-1 (HIV-1) infection. HIV-1 directly and indirectly modulates DC function to hinder the formation of effective antiviral immunity and fuel immune activation. This review focuses upon the differential dysregulation of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) at various stages of HIV-1 infection providing insights into pathogenesis. HIV-1 evades innate immune sensing by mDCs resulting in suboptimal maturation, lending to poor generation of antiviral adaptive responses and contributing to T-regulatory cell (Treg) development. Dependent upon the stage of HIV-1 infection, mDC function is altered in response to Toll-like receptor ligands, which further hinders adaptive immunity and limits feasibility of therapeutic vaccine strategies. pDC interactions with HIV-1 are pleotropic, modulating immune responses on an axis between immunostimulatory and immunosuppressive. pDCs promote immune activation through an altered phenotype of persistent type I interferon secretion and weak antigen presentation capacity. Conversely, HIV-1 stimulates secretion of indolemine 2,3 dioxygenase (IDO) by pDCs resulting in Treg induction. An improved understanding of the roles and underlying mechanisms of DC dysfunction will be valuable to the development of therapeutics to enhance HIV-specific adaptive responses and to dampen immune activation.     

Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T‐cell stimulation in response to bacille Calmette‐Guérin

To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette‐Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA‐1⁺ myeloid DCs were more susceptible than BDCA‐3⁺ mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG‐infected BDCA‐1⁺ mDCs to upregulate maturation markers and to produce granzyme B, but not IFN‐α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL‐1β availability. The synergy between the two DC subsets promoted BCG‐specific CD8⁺ T‐cell stimulation and the role of BCG‐infected BDCA‐1⁺ mDCs could not be efficiently replaced by infected BDCA‐3⁺ mDCs in the crosstalk with pDCs. We conclude that mDC–pDC crosstalk should be exploited for rational design of next‐generation TB vaccines.     

Recombinant adenovirus-transduced human dendritic cells engineered to secrete interleukin-10 (IL-10) suppress Th1-type responses while selectively activating IL-10-producing CD4+ T cells

Recombinant adenoviruses (rAd) are efficient tools for genetic modification of human dendritic cells (DC) in vitro. Infection of DCs by rAd encoding beta-galactosidase (betagal) results in partial maturation of DCs, as witnessed by the upregulation of major histocompatibility complex and costimulatory molecules. Accordingly, these DCs are more potent stimulators of Th1-type proliferative responses. We now demonstrate that infection of immature DCs with rAd encoding human interleukin (IL)-10 results in the secretion by the DCs of large amounts of IL-10, while not affecting expression of activation markers indicative of partial DC maturation. In contrast to rAd-betagal-infected DCs, rAdIL-10-infected DCs are very poor stimulators of monoclonal and polyclonal Th1-type responses. Instead, stimulation of nonpolarized CD4+ T-cell cultures with rAdIL-10-infected DCs selectively activates and expands an IL-10-producing CD4+ T-cell subset capable of suppressing Th1 responses in vitro. Our data argue that rAd-infected human DCs genetically engineered to produce IL-10 may be exploited for the modulation of harmful Th1-type responses in transplantation and autoimmune diseases.     

MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells

Major histocompatibility complex (MHC) class II-restricted antigen presentation is essential for the function of dendritic cells (DCs). We show here that plasmacytoid DCs (pDCs) differ from all other DC subsets with respect to expression of CIITA, the 'master regulator' of MHC class II genes. The gene encoding CIITA is controlled by three cell type-specific promoters: pI, pIII and pIV. With gene targeting in mice, we demonstrate that pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI. The molecular mechanisms driving MHC class II expression in pDCs are thus akin to those operating in lymphoid rather than myeloid cells.     

Generation and maturation of bone marrow-derived DCs under serum-free conditions

Standard protocols for the generation of murine dendritic cells (DCs) employ medium supplemented with heat-inactivated fetal calf serum (FCS). Recently, several attempts have been made to avoid serum exposure during DC culture. The impetus for these efforts has been a desire to generate DCs for clinical use, as preclinical data have demonstrated their efficacy in immune activation and in immune suppression both in vitro and in vivo. However, these protocols have resulted in contradictory outcomes with respect to DC survival in culture and activation status. In this report, we compared several serum-free culture conditions with respect to survival, differentiation, activation, and cytokine profile of murine DC progenitors. DC progenitors can survive only in some serum-free conditions. Surprisingly, DCs grown in serum-free medium display a higher expression of activation markers upon stimulation. They produce increased IL-12 and decreased IL-6 following stimulation. Furthermore, DCs derived under serum-free conditions may express unusual surface markers, B220 and Ly6C/G, implying an increased differentiation to plasmacytoid DCs (pDCs).     

Differential impact of late-stage HIV-1 infection on in vitro and in vivo maturation of myeloid dendritic cells

The functional and phenotypic maturation of myeloid dendritic cells (DCs), circulating and monocyte-derived, from subjects at different stages of HIV-1 infection was evaluated. The results showed that the capacity of circulating DCs was significantly impaired in subjects with CD4+ T cell counts of <200/microL, correlated with the potential of CD40 ligand expression on CD4+ T cells (R = 0.84; P = 0.002), and improved with successful antiretroviral therapy. However, the function and phenotype of monocyte-derived DCs generated by in vitro culture from subjects at any stage of HIV-1 infection were similar to those in uninfected healthy subjects. Our findings suggest that although the potential of myeloid DC precursors to achieve full maturation is preserved in subjects with late-stage HIV-1 infection, in vivo maturation of myeloid DCs was impaired in these subjects, which may be due to decreased potential of CD40 ligand expression on CD4+ T cells. That myeloid DCs fail to achieve full maturation in vivo in late-stage HIV-1 infection may contribute to the failure to induce effective cellular immunity against HIV-1 and opportunistic pathogens.     

Effective B cell activation in vitro during viremic HIV-1 infection with surrogate T cell stimulation

Identifying HIV-1-associated B cell defects and responses to activation may direct interventions to circumvent their impaired antibody responses to infection and vaccines.Among 34 viremic HIV-1-infected and 20 seronegative control adults, we measured baseline frequencies and activation of B and T cell subsets, expression of activation-induced cytidine deaminase (AID), potential determinants of B cell activation in vivo and B and T cell responses in vitro.At baseline, HIV-1 infection was associated with increased IgM memory and decreased anergic cell frequencies, as well as increased activation in all 10 B cell subsets compared with controls. HIV-1 status, T_FH activation, and BAFF were significant potential drivers of B cell activation. Despite high baseline activation among HIV-1-infected subjects, stimulation in vitro with combined surrogates for antigen (anti-IgM), cognate (CD40 ligand) and soluble T cell factors (IL-4) elicited comparable B cell activation, transitions from naïve to class-switched memory cells and AID expression in both groups.In summary, viremic HIV-1 infection perturbs circulating B cell subsets and activation at each stage of B cell maturation. However, that appropriate stimulation of B cells elicits effective activation and maturation provides impetus for advancing vaccine development to prevent secondary infections by circumventing early B cell defects.     

The microstructure of secondary lymphoid organs that support immune cell trafficking

Immune cell trafficking in the secondary lymphoid organs is crucial for an effective immune response. Recirculating T cells constantly patrol not only secondary lymphoid organs but also the whole peripheral organs. Thoracic duct lymphocytes represent an ideal cell source for analyzing T cell trafficking: high endothelial venules (HEVs) allow recirculating lymphocytes to transmigrate from the blood directly, and recirculating T cells form a cluster with dendritic cells (DCs) to survey antigen invasions even in a steady state. This cluster becomes an actual site for the antigen presentation when DCs have captured antigens. On activation, effector and memory T cells differentiate into several subsets that have different trafficking molecules and patterns. DCs also migrate actively in a manner depending upon their maturational stages. Danger signals induce the recruitment of several DC precursor subsets with different trafficking patterns and functions. In this review, we describe general and specialized structures of the secondary lymphoid organs for the trafficking of T cells and DCs by a multicolor immunoenzyme staining technique. The lymph nodes, spleen, and Peyer's patches of rats were selected as the major representatives. In vivo trafficking of subsets of T cells and DCs within these organs under steady or emergency states are shown and discussed, and unsolved questions and future prospects are also considered.     

Deciphering the message broadcast by tumor-infiltrating dendritic cells

Human dendritic cells (DCs) infiltrate solid tumors, but this infiltration occurs in favorable and unfavorable disease prognoses. The statistical inference is that tumor-infiltrating DCs (TIDCs) play no conclusive role in predicting disease progression. This is remarkable because DCs are highly specialized antigen-presenting cells linking innate and adaptive immunity. DCs either boost the immune system (enhancing immunity) or dampen it (leading to tolerance). This dual effect explains the dual outcomes of cancer progression. The reverse functional characteristics of DCs depend on their maturation status. This review elaborates on the markers used to detect DCs in tumors. In many cases, the identification of DCs in human cancers relies on staining for S-100 and CD1a. These two markers are mainly expressed by Langerhans cells, which are one of several functionally different DC subsets. The activation status of DCs is based on the expression of CD83, DC-SIGN, and DC-LAMP, which are nonspecific markers of DC maturation. The detection of TIDCs has not kept pace with the increased knowledge about the identification of DC subsets and their maturation status. Therefore, it is difficult to draw a conclusion about the performance of DCs in tumors. We suggest a novel selection of markers to distinguish human DC subsets and maturation states. The use of these biomarkers will be of pivotal importance to scrutinize the prognostic significance of TIDCs.     

Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines

During the past decade, the immunotherapeutic potential of ex vivo generated professional antigen presenting dendritic cells (DCs) has been explored in the clinic. Albeit safe, clinical results have thus far been limited. A major disadvantage of current cell-based dendritic cell (DC) therapies, preventing universal implementation of this form of immunotherapy, is the requirement that vaccines need to be tailor made for each individual. Targeted delivery of antigens to DC surface receptors in vivo would circumvent this laborious and expensive ex vivo culturing steps involved with these cell-based therapies. In addition, the opportunity to target natural and often rare DC subsets in vivo might have advantages over loading more artificial ex vivo cultured DCs. Preclinical studies show targeting antigens to DCs effectively induces humoral responses, while cellular responses are induced provided a DC maturation or activation stimulus is co-administered. Here, we discuss strategies to target antigens to distinct DC subsets and to simultaneously employ adjuvants to activate these cells to induce immunity.