DC are key regulators of immunity in view of the fact that they are involved in immune responses against infectious diseases, allergy and cancer [1, 2]. Ralph Steinman was awarded the Nobel Prize for Medicine 2011 for DC discovery in 1973 . Steinman and Cohn  described a novel cell type in mouse spleen, which they named ´dendritic cell´ due to their tree-like shape. The major function of DC is the induction of adaptive immunity in the LN.Yet, DC can also interact with innate immune cells, for instance natural killer (NK) and NKT cells [1, 4].
Upon entry of HIV into the host, the virus has to be transported from mucosal surfaces to lymphatic tissues, where it is transmitted to its primary targets, CD4+ T lymphocytes. This process is thought to be contrived by DC.
DC thereby play critical roles during HIV and SIV (simian immunodeficiency virus) infection.
The skin and mucosa are composed of two compartments, the epidermis and the dermis (skin) or stratified squamous epithelium and lamina propria (mucosa), each containing a major subset of DC - Langerhans cells (LC) reside in the suprabasal layers of the epidermis and epithelia , while dermal/interstitial DC are distributed throughout the connective tissue of the dermis [6, 7].
Both subsets represent immature DC that are very efficient in Ag uptake and processing. As immature DC (iDC), they reside in peripheral tissue, which they survey for invading pathogens. Upon encounter with antigen (Ag), DC mature (mDC) and migrate to the draining lymph nodes (LN). They pass through different maturation stages, which enable them to fulfill specific tasks such as the uptake, the processing and the presentation of Ag on major histocompatiblity complex (MHC) molecules to naïve T cells. In the T cell area of lymphatic tissue the mature DC stimulate Ag-specific CD4+ and CD8+ T cells to proliferate and develop effector function, such as cytokine production and cytotoxic activity. Effector T cells are recruited to inflamed peripheral tissue and participate in the elimination of pathogens and infected cells. This very particular life cycle illustrates why DC are called the ´sentinels of the immune system´ .
In humans, different DC subsets have been identified in blood, spleen and skin, but little is known respecting resident and migratory DC in human LN. This book chapter will review the major DC subsets found in humans and their role in HIV-pathogenesis. If data are available, also the role of the viral opsonization pattern and its impact on DC interaction will be discussed.
2. DC subsets and their role in HIV infection
DC are divided into two main groups: conventional myeloid DC (cDC) and non-conventional plasmacytoid DC (pDC) (Figure 1). As recently described byDoulatov et al. (2010) , human multi-lymphoid progenitors can bring forth all lymphoid cell types, including monocytes, macrophages and DC. Nonetheless, most DC in steady-state emerge from a common myeloid progenitor . DC areheterogenous subtypes with distinct functions, properties and localization . DC progenitors migrate from the bone-marrow through the blood to lymphoid organs and peripheral tissues. There, they give rise to different cDC subsets (Figure 1). LC display an exception within the cDC group since they maintain in the epidermis independent on circulating precursors . Within cDC, migratory and lymphoid-resident DC are distinguished: migratory DC travel from peripheral tissues to lymphoid organs, whereas lymphoid-resident DC populate lymphoid organs during their whole life-span and lack the migratory function. In humans cDC comprise Langerhans Cells (LC), dermal DC (CD103+ and CD103-), BDCA1+ (CD1c)- and BDCA3+ (CD141) DC, and the recently described CD56+ DC (Figure 1). They are localized in the skin, secondary lymphoid organs (spleen, tonsils) and blood. pDC develop in the bone-marrow and then they reside in lymphoid organs . HLA-DR+CD123+pDC express BDCA2 and this cell subset is found in blood, secondary lymphoid organs as well as peripheral tissues, e.g. skin or lungs (Figure 1). The cDC subtypes and pDC express a different receptor repertoir and comprise distinct functions with respect to HIV spread, antiviral activity and transmission, which is reviewed below and shown in Figure 1 (Table adapted from Altfeld et al., ). Both cell types are resident in lymphoid tissues in the steady state, but during an inflammatory response, pDC and cDC are actively recruited to these tissues [15-17].
2.1.1. LC and HIV
LC survey the basal and suprabasal layers of the stratified squamous epithelium of the skin and oral and ano-genital mucosa for invading pathogens [18-21]. Due to their ideal localization in mucosal tissues and their long dendrites to efficiently capture Ag, they comprise the first line defense against mucosal infections. After Ag acquisition, LC start to mature, as represented by up-regulation of co-stimulatory molecules (CD80, CD86, CD40), MHC class I and II molecules, CD83 and CCR7 and down-regulation of Langerin and E-cadherin .
Due to CCR7 up-regulation, the mature LC migrate to the LN along a CCL19 and CCL21-leu (leucine isoform of CCL21) gradient to efficiently prime T cells there . Beside initiating an effective adaptive immune response, LC were illustrated by DeWitte et al. [24, 25] to also have important functions with respect to innate immune responses. Beside a specific set of TLRs (TLR2, 3, 5) and high expression of CD1a, LC express Langerin and contain Birbeck granules that might be crucial to their innate function [21, 26-29] (Figure 1). The C-type lectin Langerin interacts with non-opsonized HIV-1 (Figure 2) and other pathogens such as fungi and bacteria, via fucose or mannose residues. Thereby degradation of HIV-1 in Birbeck granules is promoted and HIV-1 dissemination is limited .
Early investigations of HIV-LC interactions illustrated that LC are productively infected by HIV and that they efficiently transmit the virus to T cells [30-32]. These results suggested that HIV take advantage of the antigen-capturing properties of LC to reach the T cell zone in the lymphatic tissues and via this route, HIV can establish a productive infection of the host. However,
Engagement of Langerin trimers on the surface of LC induces formation of Birbeck granules, which are part of the endosomal recycling system and uniquely found in LC (Figure 3, lower right panel) . Upon capture of mycobacterial lipoproteins by Langerin, these were exposed to CD1a molecules in Birbeck granules , which suggests that Birbeck granule formation displays a non-classical antigen processing pathway . Also HIV-1 attaches to Langerin on LC and is subsequently routed to Birbeck granules, which points to a role of the granules with respect to degradation of viruses.
Studies by Gejitenbeek´s laboratory  showed that under homeostatic conditions, Langerin expressed on LC and acts as restriction factor for HIV infection. They demonstrated that, if HIV-gp120 attaches to Langerin, the viral particle is internalized and subsequently degraded in Birbeck granules. Thus, LC are protected from infection with incoming, non-opsonized HIV particles and HIV-1 is not disseminated throughout the host . The rapid internalization of HIV-1 into LC by Langerin impedes interactions and subsequent fusion with CD4 and CCR5 and also prevents transmission to the main target cells of the virus, CD4+ T cells. Thereby, Langerin acts as a protective anti-HIV barrier during the first steps of HIV-1 infection, if the virus is non-opsonized and if sexually transmitted pathogens are lacking.
As demonstrated, if the host system is facing other sexually transmitted infections, the anti-HIV-1-barrier of LC is abrogated and HIV-1 transfer to susceptible CD4+ T cells is promoted [20, 36, 37]. Pathogens, such as Candida or Neisseria, directly interact with Langerin and compete with HIV-1-binding. Additional factors explaining the by-passing of the anti-HIV-1-barrier function of Langerin are that:
by high viral loads the receptor becomes saturated,
infections, e.g. Herpes simplex virus infection, down-regulate Langerin surface expression ,
the HIV-1 entry receptors CD4 and CCR5 are up-regulated during additional sexually transmitted infections ,
or the antiviral function of Langerin is reverted by inflammation-induced TNF-α (tumor-necrosis-factor α) production due to Candida albicans or Neisseria gonorrhoea .
These observations allow to conclude that during acute co-infection the anti-viral function of LC is significantly decreased.
Not only acute co-infection, but also opsonization of HIV with either complement fragments or specific Abs might result in reduction or abolishment of the anti-viral function mediated by Langerin (
In summary, during acute co-infection or by opsonization with complement fragments or Abs, the anti-viral function of LC is significantly reduced due to competition for Langerin or different receptor utilization. This facilitates HIV-1 infection of LC via CD4 and CCR5, intracellular uptake of the virus (Figure 3) and promotion of HIV-1 transfer to its targets, CD4+ T cells.
On the other hand LC were implicated in establishment of infection due to their location in the foreskin and due to compelling evidence that male circumcision efficiently reduces the risk to become infected with HIV-1 . It was furthermore shown
2.1.2. Dermal DC and HIV
Along with LC, HIV-1 firstly attaches to dermal (interstitial) DC upon entry at mucosal surfaces (Figure 1). Dermal DC are underlying the epithelium, do not contain Birbeck granules and express heterogenous amounts of CD1a .
Interstitial DC are localized in the dermis and oral, vaginal and colonic lamina propria [6, 49-52]. They are characterized by the expression of CD11c, high expression of various C-type lectin receptors (Langerin on CD103+ DC, DC-SIGN on CD103- DC, DEC-205 on both subsets), TLR2, 3, 4 and 5 and they secrete various cytokines upon pathogenic stimulation (Figure 1). Since there are only 2 studies available on human CD103+ DC and SIV [53, 54], the following chapter refers to CD103-, DC-SIGN+ dermal DC.
As shown in Figure 3, dermal DC (left panel) and LC (right panel), which emigrated from whole skin explants, take up variable amounts of HIV-1 particles. As demonstrated by Frank et al. , human and macaque DC interacted similarly with SIV and ample amounts of virus were captured by DC. Transmission electron microscopic analyses revealed that iDC, which are endocytically highly active, captured few viral particles near the periphery of the membrane, while mDC, which down-regulate the endocytic capacity, retained high amounts of virions in large vesicular compartments deeper within DC . This points to a diverse entry and handling of virions within iDC and mDC.
Beside the different handling of HIV-1 or SIV within iDC and mDC, opsonization of the virus with either complement fragments and/or Abs significantly affects the binding mechanism, internalization and infection of DC as well as their T cell stimulatory capacity [41, 42, 58]. As shown by Pruenster et al. , the complement cloud around the virus significantly blocked the accessibility of gp120 and therefore interfered with C-type lectin interaction.
Similar amounts of HIV-1 bound to the surface of DC independent on the opsonization pattern of the virus (Pruenster et al., 2005). The attachment of the differentially opsonized HIV-1-preparations was found to be specific (Figure 4 [
blocking α-DC-SIGN mAb inhibited interaction with non-opsonized HIV-1 (Figure 4, HIV),
blocking α-CR3mAb (TMG6.5) significantly interferred with binding of complement-opsonized HIV (HIV-C) to DC (Fig.4, HIV-C) and
blocking α-CD32mAb (AT10) inhibited binding of Ab-opsonized HIV-1 (Figure 4, HIV-Ig).
Additionally, we found variations respecting infection of DC with differentially opsonized HIV-1 preparations . Productive infection of DC and LC with HIV-1 was described to be relatively inefficient compared to HIV-infection of CD4+ T cells and HIV- or SIV-infected DC are rarely detected
In contrast, HIV-1 coated with specific, non-neutralizing Abs significantly impaired infection of and integration in DC and also ´
Despite the low-level productive infection of DC, non-opsonized HIV-1 is very efficiently transmitted to T cells either via de novo (´
Lastly, the antigen-presenting capacity of DC was also shown to be modulated by the opsonization pattern of the virus . Earlier studies illustrated the role of complement opsonization respecting induction of effective CTL responses against viral infections, but the exact mechanism was not determined [64-66]. The exclusive role of DC in priming naïve CD8+ T cells in response to exogenous cell-associated as well as endogenously synthesized Ags has been shown [67, 68]. Endogenously synthesized antigens from DC infected with LCMV (choriomeningitis virus) mediated strong CTL responses, while macrophages and B cells infected with LCMV did not induce CTLs .
We recently found that opsonization of retroviral particles with complement fragments enhanced the ability of DC to induce CTL responses both
Additionally, we demonstrated that in contrast to complement opsonization, antibody-coating of the viral surface attenuated the CTL-stimulatory capacity of HIV-exposed DC . In some HIV-1-positive individuals, high levels of antibodies and low levels of complement fragments coat the HIV-1 surface and therefore we investigated the effects of the non-neutralizing Abs bound to the surface of HIV-1 on the CTL-stimulatory capacity of DC. We observed
Preferential expression of CCR5 on immature LC and DC restricts the transmission of X4-tropic isolates at the site of infection. Additionally,
2.1.3. Blood DC and HIV
18.104.22.168. BDCA1+ DC, BDCA3+ DC, CD56+ DC and HIV
BDCA1+ myeloid DC can be directly isolated from human blood. This population was described to be reduced in the blood of HIV-infected individuals [76-78]. We found that BDCA1+ DC exerted a decreased transmission of HIV-1 to autologous CD4+ T cells, when the virus was opsonized with specific IgGs in contrast to non- or complement-opsonized HIV-1 and when the T cells were added delayed . When CD4+ T cells were immediately added after washing the differentially loaded DC, the same infection efficiency was observed using HIV, HIV-C or HIV-Ig . The two-phase transfer of HIV to DC as described above (
BDCA3+ DC represent the human equivalent to mouse CD8α+ DC and they are the major producers of IFN-λ in response to dsRNA poly I:C .As recently described by Dutertre et al.  using an 11-color flow cytometric strategy, circulating BDCA1+ DC and BDCA3+ DC counts were reduced in 15 viremic, untreated patients compared to 8 HIV-1-positive individuals under treatment and 13 healthy donors. By using this method, they illustrated that both blood DC subsets expressed characteristic lineage markers: BDCA1+ DC expressed CD14, while particularly BDCA3+ DC displayed CD56 on their surface. BDCA3+ DC were shown to be more significantly down-modulated in viremic patients compared to controls  and it remains to be investigated by longitudinal studies, if combined antiretroviral therapy can restore the pool of circulating myeloid BDCA1+ and BDCA3+ DC.
Blood CD56+ DC were recently described by Gruenbacher et al.  and comprise intermediate-sized lymphocytes with an HLA-DRhigh, CD80+ and CD86+ expression profile. Upon cultivation they acquire DC-like morphology with increased levels of above mentioned surface markers. Upon stimulation, they are able to efficiently stimulate CD56+γδ T cells, which results in secretion of IFNγ, TNF-α, and IL-1β . The role of CD56+ DC respecting HIV-1 infection and pathogenesis needs to be further investigated.
2.2. pDC and HIV
Plasmacytoid DC (pDC) (Figure 1) or type 1 IFN-producing dendritic cells are innate immune cells in blood, which are specialized in releasing massive amounts of IFNα and IFNβ upon viral challenge, including HIV-1 . They constitute <0.2-0.5% of peripheral blood mononuclear cells (PBMC)  and in humans, pDC express the characteristic surface markers BDCA-2 (CD303, CLEC4C) and CD123 along with BDCA4 (CD304, NRP1), but they do not express CD11c, a marker of myeloid DC, or CD14 .
pDCs are key players of the innate immune response
Data by Zhou et al.  indicate that subsequent to HIV-1 challenge, signaling via TLR7 triggers autophagy and increased IFNα production from human pDC. The IFNα secretion mediated by an autophagy-dependent pathway may play an important role for T cell triggering during HIV-1 pathogenesis.
Beside acting as pro-inflammatory cells, pDC also provide negative regulatory signals and thus induce tolerance. pDC express IDO (indoleamine 2,3-dioxygenase) and PDL-1 (programmed death ligand; 1) which are associated with the negative modulation of T cell responses and regulatory T cell induction [100-102].
During acute HIV-1 infection, NK cells are recruited and activated by pDC to the sites of infection and to LN due to IFNα secretion [103, 104]. IFNα was demonstrated to increase the perforin levels in NK and CD8+ T cells. At the sites of infection ´DC-editing´ occurs by NK cells, since activated NK cells delete immature pDC to select for the more immunogenic mature pDC [105-108].Beside NK cell recruitment and activation, pDC-secreted IFNα promotes maturation and migration of other DC subsets. Due to their localization, it is unlikely that pDC are involved in HIV-1 capture, transport and transmission, but they are supposed to control HIV-1 in the acute phase of infection due to their immediate antiviral and NK priming activity.
Chronic exposure to HIV-1 leads to hyperactivation of pDC resulting in simultaneous type I IFN secretion and IDO expression. Thus, pDC concurrently exert cytotoxic and suppressive effects on T cells during chronic HIV-1 infection .
HIV-1 infection not only disrupts DC homeostasis within myeloid DC subsets, but also pDC homeostasis is defective during chronic HIV-1 infection. cDC and pDC are lost from blood, which correlates with high viral loads and low CD4+ T cell counts [76, 110-113]. Deficiencies in pDC function were among the earliest observations of immune dysfunction in HIV-1 infection and some of the earliest studies of the ´natural IFN-α-producing cells´ (i.e. pDC) illustrated that PBMC from AIDS patients were severely compromised in their ability to produce IFN-α
Cell death and/or a failure of bone marrow progenitors to differentiate into pDC might contribute to the loss of pDC from blood of chronically infected individuals. In non-pathogenic models of SIV infection, no depletion of blood pDC was observed [114, 115] and HIV-1-positive individuals, who are able to control infection (= long-term non-progressors) were also shown to have increased numbers of blood pDC .In contrast, it was described that during HIV-2 infection, which is highly attenuated compared to HIV-1 infection in humans, also the numbers of blood pDC is found reduced . Thereby, the exact role of pDC depletion during HIV infection is not clear yet.
The depletion of cDC from the sites of infection was ascribed to a higher expression of CCR7 on the surface of cDC and a signficantly increased CCL19 expression in LN of SIV-infected animals, thus suggesting that inflamed LN lure cDC away from the sites of infection early during progressive SIV infection . A similar mechanism can be imagined for pDC, which are recruited to inflamed LN via CXCL9 and E-selectin [16, 118], but the pDC loss could also be due to direct infection, enhanced apoptosis or CD95 up-regulation [119-123].
Not only pDC numbers are decreased during on-going HIV-1 infection, but also the quality of the cells is suffering. They exert a reduced ability to migrate towards the CXCR4 ligand CXCL12 , they stimulate Treg cells to dampen HIV-1 immunity and they furthermore shift the Treg-Th17 balance [125, 126]. So far, interactions of differentially opsonized HIV-1 preparations with pDC has not been investigated.
3. Outlook: Impact of the HIV-1 opsonization pattern on DC function
As follows of investigations on HIV-1 in the last 30 years, antibody responses against the virus are not effective and cellular immune responses not powerful enough to suppress or even control HIV-1. DC, the prime inducers and regulators of immunity and tolerance, are crucial in designing modern vaccines [127-130]. Therefore, nowadays vaccine science shall combine established classical vaccine approaches with new attempts based on the expanded immunological knowledge.
Innate and adaptive immune responses are needed to generate efficient, long-lasting protection. Immediate innate responses involve activation of the complement system, ligation of pattern recognition receptors e.g. TLRs, C-type lectins, activation of NK cells, cDC and pDC, and type I, II, as well as III interferons. For viral clearance, the optimal balance between CD4+ and CD8+ T cells is required during the adaptive immune responses. Current HIV-1 vaccination strategies include the use of peptides or monocyte-derived DC exposed to chemically inactivated HIV-1 and aim in designing a vaccine efficiently inducing both, cellular and humoral immune responses [131-133]. So far, disappointing results have been achieved in clinical trials targeting either cellular [134, 135] or humoral immunity [136, 137]. The most prominent AIDS vaccine trial so far was the RV144 in Thailand , which evoked strong, but transient Env-specific CD4+ T cell and Ab responses, but only weak HIV-specific CD8+ T cell responses [131, 138].
HIV-1 induces immediate responses of the immune system upon entering mucosal surfaces. There, the complement system constitutes a first line of defense against the virus. Recently, we illustrated an important role for complement opsonization of retroviruses as an endogenous adjuvant for DC-mediated CTL-induction .
Efficient early CD8+ T cell responses are crucial in controlling HIV-1 replication and their key role in HIV-1 control is additionally substantiated by association of certain HLA class I alleles and an improved disease progression [139-141]. In view of our very recent observations (, ), we propose that CD8+ T cells are efficiently primed by DC during acute viral infection, particularly by enhanced infection of DC with HIV-C . Thus, more efficient presentation of endogenously synthesized viral Ags via HLA-ABC , and ore efficient cross-presentation from incoming complement-opsonized HIV-1 are mediated. In contrast, Ab-opsonization of HIV-1 weakens the CTL-induction by modulation of DC function and might influence future vaccination strategies .
As shown by Lu et al. [142-144]
LC were described to allow more cross-priming of CD8+ T cells, while dermal DC are more specialized in primingnaive CD4+ T cells . The finding that complement-opsonization of HIV prior loading of DC significantly enhanced the CD8+ T cell-stimulatory capacity of the cells in combination with using specific DC subtypes might efficiently improve future vaccination strategies and there is good reason to address DC of the skin, especially Langerhans cells, for purposes of vaccination.
A greater understanding of the innate and adaptive processes and the different functions of DC subsets to HIV-1 infection will lead to development of an effective vaccine.
The authors would like to thank Nikolaus Romani and Hella Stössl for providing the transmission electron microscopic picture.
The work of the authors is supported by the Austrian Science Fund [FWF, P22165 and P24598 to DW], the Tyrolean Science Fund [TWF, project: D-155140-016-011 to WP] and the OeNB [project: 14875 to WP].