Teunis B. H. Geijtenbeek

Signalling through C-type lectin receptors: shaping immune responses

C-type lectin receptors (CLRs) expressed by dendritic cells are crucial for tailoring immune responses to pathogens. Following pathogen binding, CLRs trigger distinct signalling pathways that induce the expression of specific cytokines which determine T cell polarization fates. Some CLRs can induce signalling pathways that directly activate nuclear factor-κB, whereas other CLRs affect signalling by Toll-like receptors. Dissecting these signalling pathways and their effects on host immune cells is essential to understand the molecular mechanisms involved in the induction of adaptive immune responses. In this Review we describe the role of CLR signalling in regulating adaptive immunity and immunopathogenesis and discuss how this knowledge can be harnessed for the development of innovative vaccination approaches.

Langerin is a natural barrier to HIV-1 transmission by Langerhans cells.

Human immunodeficiency virus-1 (HIV-1) is primarily transmitted sexually. Dendritic cells (DCs) in the subepithelium transmit HIV-1 to T cells through the C-type lectin DC-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN). However, the epithelial Langerhans cells (LCs) are the first DC subset to encounter HIV-1. It has generally been assumed that LCs mediate the transmission of HIV-1 to T cells through the C-type lectin Langerin, similarly to transmission by DC-SIGN on dendritic cells (DCs). Here we show that in stark contrast to DC-SIGN, Langerin prevents HIV-1 transmission by LCs. HIV-1 captured by Langerin was internalized into Birbeck granules and degraded. Langerin inhibited LC infection and this mechanism kept LCs refractory to HIV-1 transmission; inhibition of Langerin allowed LC infection and subsequent HIV-1 transmission. Notably, LCs also inhibited T-cell infection by viral clearance through Langerin. Thus Langerin is a natural barrier to HIV-1 infection, and strategies to combat infection must enhance, preserve or, at the very least, not interfere with Langerin expression and function.

Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1β via a noncanonical caspase-8 inflammasome

Production of the proinflammatory cytokine interleukin 1β (IL-1β) by dendritic cells is crucial in host defense. Here we identify a previously unknown role for dectin-1 in the activation of a noncanonical caspase-8 inflammasome in response to fungi and mycobacteria. Dectin-1 induced both the production and maturation of IL-1β through signaling routes mediated by the kinase Syk. Whereas the CARD9–Bcl-10–MALT1 scaffold directed IL1B transcription, the recruitment of MALT1–caspase-8 and ASC into this scaffold was crucial for processing of pro-IL-1β by caspase-8. In contrast to activation of the canonical caspase-1 inflammasome, which requires additional activation of cytosolic receptors, activation of the noncanonical caspase-8 inflammasome was independent of pathogen internalization. Thus, dectin-1 acted as an extracellular sensor for pathogens that induced both IL-1β production and maturation through a noncanonical caspase-8-dependent inflammasome for protective immunity.

Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori

Cooperation between different innate signaling pathways induced by pattern-recognition receptors (PRRs) on dendritic cells (DCs) is crucial for tailoring adaptive immunity to pathogens. Here we show that carbohydrate-specific signaling through the C-type lectin DC-SIGN tailored cytokine production in response to distinct pathogens. DC-SIGN was constitutively associated with a signalosome complex consisting of the scaffold proteins LSP1, KSR1 and CNK and the kinase Raf-1. Mannose-expressing Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) induced the recruitment of effector proteins to the DC-SIGN signalosome to activate Raf-1, whereas fucose-expressing pathogens such as Helicobacter pylori actively dissociated the KSR1–CNK–Raf-1 complex from the DC-SIGN signalosome. This dynamic regulation of the signalosome by mannose- and fucose-expressing pathogens led to the enhancement or suppression of proinflammatory responses, respectively. Our study reveals another level of plasticity in tailoring adaptive immunity to pathogens.

Unique appearance of proliferating antigen-presenting cells expressing DC-SIGN (CD209) in the decidua of early human pregnancy.

Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83(+) DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR(+)/DC-SIGN(+) APCs further phenotyped as CD14(+)/CD4(+)/CD68(+/-)/CD83(-)/CD25(-). These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209(+) cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN(+) decidual APCs were observed in situ to have intimate contact with CD56(+)/CD16(-)/ICAM-3(+) decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14(+)/DC-SIGN(+) decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14(+)/DC-SIGN(+) decidual cells into CD25(+)/CD83(+) mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset.

HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells

Pattern-recognition receptors (PRRs) elicit antiviral immune responses to human immunodeficiency virus type 1 (HIV-1). Here we show that HIV-1 required signaling by the PRRs Toll-like receptor 8 (TLR8) and DC-SIGN for replication in dendritic cells (DCs). HIV-1 activated the transcription factor NF-κB through TLR8 to initiate the transcription of integrated provirus by RNA polymerase II (RNAPII). However, DC-SIGN signaling was required for the generation of full-length viral transcripts. Binding of the HIV-1 envelope glycoprotein gp120 to DC-SIGN induced kinase Raf-1–dependent phosphorylation of the NF-κB subunit p65 at Ser276, which recruited the transcription-elongation factor pTEF-b to nascent transcripts. Transcription elongation and generation of full-length viral transcripts was dependent on pTEF-b-mediated phosphorylation of RNAPII at Ser2. Inhibition of either pathway abrogated replication and prevented HIV-1 transmission. Thus, HIV-1 subverts crucial components of the immune system for replication that might be targeted to prevent infection and dissemination.

Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques.

Measles virus (MV) is hypothesized to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (CD150), which has been identified as the receptor for wild-type MV. We have infected rhesus and cynomolgus macaques with a recombinant MV strain expressing enhanced green fluorescent protein (EGFP); thus bringing together the optimal animal model for measles and a virus that can be detected with unprecedented sensitivity. Blood samples and broncho-alveolar lavages were collected every 3 d, and necropsies were performed upon euthanasia 9 or 15 d after infection. EGFP production by MV-infected cells was visualized macroscopically, in both living and sacrificed animals, and microscopically by confocal microscopy and FACS analysis. At the peak of viremia, EGFP fluorescence was detected in skin, respiratory and digestive tract, but most intensely in all lymphoid tissues. B- and T-lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to 30%) of infected lymphocytes were detected in lymphoid tissues, and the virus preferentially targeted cells with a memory phenotype. Unexpectedly, circulating monocytes did not sustain productive MV infection. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells were detected in conjunction with infected T-lymphocytes, suggesting transmission of MV between these cell types. Fluorescent imaging of MV infection in non-human primates demonstrated a crucial role for lymphocytes and dendritic cells in the pathogenesis of measles and measles-associated immunosuppression.

Early Target Cells of Measles Virus after Aerosol Infection of Non-Human Primates

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMVKSEGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n = 3 per time point) and infected (EGFP+) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP+ cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

Selective C-Rel Activation via Malt1 Controls Anti-Fungal T-H-17 Immunity by Dectin-1 and Dectin-2

C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of TH1 and TH-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to TH-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of TH-17-polarizing cytokines IL-1β and IL-23p19. Malt1 inhibition abrogates c-Rel activation and TH-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of TH-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized TH-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive TH-17 immunity to fungi via Malt1-dependent activation of c-Rel.

Innate signaling by the C-type lectin DC-SIGN dictates immune responses

Effective immune responses depend on the recognition of pathogens by dendritic cells (DCs) through pattern recognition receptors (PRRs). These receptors induce specific signaling pathways that lead to the induction of immune responses against the pathogens. It is becoming evident that C-type lectins are also important PRRs. In particular, the C-type lectin DC-SIGN has emerged as a key player in the induction of immune responses against numerous pathogens by modulating TLR-induced activation. Recent reports have begun to elucidate the molecular mechanisms underlying these immune responses. Upon pathogen binding, DC-SIGN induces an intracellular signaling pathway with a central role for the serine/threonine kinase Raf-1. For several pathogens that interact with DC-SIGN, including Mycobacterium tuberculosis and HIV-1, Raf-1 activation leads to acetylation of NF-κB subunit p65, which induces specific gene transcription profiles. In addition, other DC-SIGN-ligands induce different signaling pathways downstream of Raf-1, indicating that DC-SIGN-signaling is tailored to the pathogen. In this review we will discuss in detail the current knowledge about DC-SIGN signaling and its implications on immunity.