Indolamine 2,3‐dioxygenase expression by monocytes and dendritic cell populations in hepatitis C patients

Dendritic cells (DCs) play an important role in the induction of the primary immune response to infection. DCs may express the tryptophan‐catabolizing enzyme indolamine2,3‐dioxygenase (IDO), which is an inducer of immune tolerance. Because there is evidence that chronic hepatitis C virus (HCV) infection leads to functional impairment of certain DC populations, we analysed IDO expression in DCs and monocytes from chronically infected and recovered HCV patients. The IDO1 and ‐2 expression was increased significantly in the monocytes of chronic HCV patients but, interestingly, not in those from recovered patients. The myeloid DCs from chronically infected HCV patients also showed enhanced IDO1 expression, while no change in either IDO1 or ‐2 was found for plasmacytoid DCs. Up‐regulation of IDO1 gene expression was confirmed by the presence of enhanced kynurenine/tryptophan ratios in the plasma from chronic HCV patients. Increased IDO1 and ‐2 expression was also observed in monocytes from healthy donors infected with an adapted mutant of the HCV JFH‐1 strain ex vivo, confirming a direct effect of HCV infection. These changes in IDO expression could be prevented by treatment with the IDO inhibitor 1‐methyl tryptophan (1‐mT). Furthermore, maturation of monocyte‐derived DCs from chronically infected HCV patients, as well as well as monocyte‐derived DCs infected ex vivo with HCV, was impaired, but this was reversed by 1‐mT treatment. This suggests that IDO inhibitors may be used to treat chronic HCV patients in vivo, in conjunction with current therapies, or to activate DCs from patients ex vivo, such that they can be administered back as a DC‐based therapeutic vaccine.     

In vivo targeting of human DC‐SIGN drastically enhances CD8+ T‐cell‐mediated protective immunity

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T‐cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC‐SIGN (DC‐specific‐ICAM3‐grabbing‐nonintegrin/CD209) is a C‐type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC‐SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC‐SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti‐DC‐SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti‐DC‐SIGN antibodies conjugated to OVA induced strong and persistent antigen‐specific CD4⁺ and CD8⁺ T‐cell responses, which efficiently protected from infection with OVA‐expressing Listeria monocytogenes. Thus, we propose DC targeting via DC‐SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.     

The CD8+ dendritic cell subset

Summary: Mouse lymphoid tissues contain a subset of dendritic cells (DCs) expressing CD8α together with a pattern of other surface molecules that distinguishes them from other DCs. These molecules include particular Toll-like receptor and C-type lectin pattern recognition receptors. A similar DC subset, although lacking CD8 expression, exists in humans. The mouse CD8⁺ DCs are non-migrating resident DCs derived from a precursor, distinct from monocytes, that continuously seeds the lymphoid organs from bone marrow. They differ in several key functions from their CD8⁻ DC neighbors. They efficiently cross-present exogenous cell-bound and soluble antigens on major histocompatibility complex class I. On activation, they are major producers of interleukin-12 and stimulate inflammatory responses. In steady state, they have immune regulatory properties and help maintain tolerance to self-tissues. During infection with intracellular pathogens, they become major presenters of pathogen antigens, promoting CD8⁺ T-cell responses to the invading pathogens. Targeting vaccine antigens to the CD8⁺ DCs has proved an effective way to induce cytotoxic T lymphocytes and antibody responses.     

Butyrate and retinoic acid imprint mucosal‐like dendritic cell development synergistically from bone marrow cells

Accumulating data show that the phenotypes and functions of distinctive mucosal dendritic cells (DCs) in the gut are regulated by retinoic acid (RA). Unfortunately, the exact role of butyrate in RA‐mediated mucosal DC differentiation has not been elucidated thoroughly to date. Mucosal‐like dendritic cell differentiation was completed in vitro by culturing bone marrow cells with growth factors [granulocyte–macrophage colony‐stimulating factor (GM‐CSF/interleukin (IL)‐4], RA and/or butyrate. The phenotypes, cytokine secretion, immune functions and levels of retinal dehydrogenase of different DCs were detected using quantitative polymerase chain reaction (qPCR), enzyme‐linked immunosorbent assay (ELISA) and flow cytometry, respectively. The results showed that RA‐induced DCs (RA‐DCs) showed mucosal DC properties, including expression of CD103 and gut homing receptor α₄β₇, low proinflammatory cytokine secretion and low priming capability to antigen‐specific CD4⁺ T cells. Butyrate‐treated RA‐DCs (Bu‐RA‐DCs) decreased CD11c, but increased CD103 and α₄β₇ expression. Moreover, the CD4⁺ T priming capability and the levels of retinal dehydrogenase of RA‐DCs were suppressed significantly by butyrate. Thus, butyrate and retinoic acid have different but synergistic regulatory functions on mucosal DC differentiation, indicating that immune homeostasis in the gut depends largely upon RA and butyrate to imprint different mucosal DC subsets, both individually and collectively.     

Phenotypic switch of CD8+ T cells reactivated under hypoxia toward IL‐10 secreting,poorly proliferative effector cells

CD8⁺ T cells controlling pathogens or tumors must function at sites where oxygen tension is frequently low, and never as high as under atmospheric culture conditions. However, T‐cell function in vivo is generally analyzed indirectly, or is extrapolated from in vitro studies under nonphysiologic oxygen tensions. In this study, we delineate the role of physiologic and pathologic oxygen tension in vitro during reactivation and differentiation of tumor‐specific CD8⁺ T cells. Using CD8⁺ T cells from pmel‐1 mice, we observed that the generation of CTLs under 5% O₂, which corresponds to physioxia in lymph nodes, gave rise to a higher effector signature than those generated under atmospheric oxygen fractions (21% O₂). Hypoxia (1% O₂) did not modify cytotoxicity, but decreasing O₂ tensions during CTL and CD8⁺ tumor‐infiltrating lymphocyte reactivation dose‐dependently decreased proliferation, induced secretion of the immunosuppressive cytokine IL‐10, and upregulated the expression of CD137 (4‐1BB) and CD25. Overall, our data indicate that oxygen tension is a key regulator of CD8⁺ T‐cell function and fate and suggest that IL‐10 release may be an unanticipated component of CD8⁺ T cell‐mediated immune responses in most in vivo microenvironments.     

Cytokine‐dependent regulation of dendritic cell differentiation in the splenic microenvironment

The DC‐derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady‐state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α^?CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α‐galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α^? and CD8α⁺ splenic DC subsets and enhances cross‐presentation of exogenous antigens. Based on genome‐wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN‐γ‐mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN‐γR and are much less responsive to IFN‐γ. Under inflammatory conditions, particularly in the absence of IFN‐γ signaling in IFN‐γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM‐CSF in concert with IL‐4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.     

Vaccine molecules targeting Xcr1 on cross‐presenting DCs induce protective CD8+ T‐cell responses against influenza virus

Targeting antigens to cross‐presenting dendritic cells (DCs) is a promising method for enhancing CD8⁺ T‐cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross‐presenting murine CD8α⁺ DCs, and that the expression is conserved on homologous DC subsets in humans (CD141⁺ DCs), sheep (CD26⁺ DCs), and macaques (CADM1⁺ DCs). We therefore tested if targeting antigens to Xcr1 on cross‐presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α⁺ DCs and increased proliferation of antigen‐specific T cells. DNA vaccines encoding dimeric Xcl1‐hemagglutinin (HA) fusion proteins induced cytotoxic CD8⁺ T‐cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8⁺ T‐cell responses, targeting of antigen to Xcr1 induced CD4⁺ Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α⁺ DCs using Xcl1 represents a novel and promising method for induction of protective CD8⁺ T‐cell responses.     

High Functional CD70 Expression on α‐Type 1‐Polarized Dendritic Cells from Patients with Chronic Lymphocytic Leukaemia

Antigen‐loaded dendritic cells (DCs) used as anticancer vaccine holds promise for therapy, but needs to be optimized. The most frequently described DC vaccine is being matured with a cocktail containing prostaglandin E₂ (PGE₂DC). However, even though PGE₂DCs express both costimulatory and migratory receptors, their IL‐12p70‐prodcution is low, leading to an insufficient Th1 immune response. As an alternative, α‐type‐1 polarized DCs (αDC1s) have shown a superior production of IL‐12p70 and subsequent activation of effector cells. From chronic lymphocytic leukaemia (CLL) patients, αDC1s can be generated to induce a functional Th1‐immune response. Yet, another costimulatory receptor, CD70, appears to be essential for optimal DC function by promotion of T cell survival and function. So far, PGE₂ is suggested as one of the most important factors for the induction of CD70 expression on DCs. Therefore, we wanted to investigate whether αDC1s have the ability to express functional CD70. We found that CD70 expression on αDC1s could be upregulated in the same manner as PGE₂DCs. In an allogeneic mixed leucocyte reaction, we found that antibody‐blocking of CD70 on αDC1s from controls reduced effector cell proliferation although this could not be found when using CLL αDC1s. Nevertheless, CD70‐blocking of αDC1s from both controls and patients with CLL had a negative influence on the production of both IL‐12p70 and the Th1 cytokine IFN‐γ, while the production of the Th2 cytokine IL‐5 was enhanced. Together, this study further suggests that αDC1s should be considered as a suitable candidate for clinical antitumour vaccine strategies in patients with CLL.     

Generation of Potent Cytotoxic T Lymphocytes Against Castration‐Resistant Prostate Cancer Cells by Dendritic Cells Loaded With Dying Allogeneic Prostate Cancer Cells

To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)‐based immunotherapy against prostate cancer, various tumour antigens should be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for castration‐resistant prostate cancer (CRPC) using prostate cancer–specific CTLs generated in vitro by DCs. Monocyte‐derived DCs from patients with CRPC were induced to mature using a standard cytokine cocktail (in IL‐1β, TNF‐α, IL‐6 and PGE₂: standard DCs, sDCs) or using an α‐type 1‐polarized DC (αDC1) cocktail (in IL‐1β, TNF‐α, IFN‐α, IFN‐γ and polyinosinic:polycytidylic acid) and loaded with the UVB‐irradiated CRPC cell line PC‐3. Antigen‐loaded DCs were evaluated by morphological and functional assays. The αDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the αDC1s were loaded with tumour antigens or not, compared to sDCs. The αDC1s showed a higher production of interleukin‐12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumour antigens, as compared to standard DCs (sDCs). Prostate cancer–specific CTLs against autologous CRPC cells were successfully induced by αDC1s loaded with dying PC‐3 cells. Autologous αDC1s loaded with an allogeneic CRPC cell line can generate greater CRPC‐specific CTL responses as compared to sDCs and may provide a novel source of DC‐based vaccines that can be used for the development of immunotherapy in patients with CRPC.     

GM‐CSF therapy inhibits chronic graft‐versus‐host disease via expansion of regulatory T cells

Regulatory T cells (Tregs) attenuate excessive immune responses, making their expansion beneficial in immune‐mediated diseases, including allogeneic bone marrow transplantation associated with graft‐versus‐host disease (GVHD). In addition to interleukin‐2, Tregs require T‐cell receptor and costimulatory signals from antigen‐presenting cells, such as DCs, for their optimal proliferation. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) increases DC number and may promote DC‐dependent Treg proliferation. Here, we demonstrate that GM‐CSF treatment increases CD4⁺CD8^– DCs, which are associated with Treg expansion. In a mouse model of chronic GVHD (cGVHD), GM‐CSF therapy expanded Tregs, protected against the development of skin GVHD, and regulated both Th1 and Th17 responses in the peripheral lymph nodes, resulting in an attenuation of skin cGVHD. Notably, the expanded Tregs were instrumental to GM‐CSF‐mediated cGVHD inhibition, which was dependent upon an increased ratio of Tregs to conventional T cells rather than augmentation of suppressive function. These data suggest that GM‐CSF induces Treg proliferation by expanding CD4⁺CD8^? DCs, which in turn regulate alloimmune responses in a cGVHD mouse model. Thus, GM‐CSF could be used as a therapeutic DC modulator to induce Treg expansion and to inhibit excessive alloimmune responses in immune‐related diseases.     

Virulence‐dependent induction of interleukin‐10‐producing‐tolerogenic dendritic cells by Mycobacterium tuberculosis impedes optimal T helper type 1 proliferation

Mycobacterium tuberculosis inhibits optimal T helper type 1 (Th1) responses during infection. However, the precise mechanisms by which virulent M. tuberculosis limits Th1 responses remain unclear. Here, we infected dendritic cells (DCs) with the virulent M. tuberculosis strain H37Rv or the attenuated strain H37Ra to investigate the phenotypic and functional alterations in DCs and resultant T‐cell responses. H37Rv‐infected DCs suppressed Th1 responses more strongly than H37Ra‐infected DCs. Interestingly, H37Rv, but not H37Ra, impaired DC surface molecule expression (CD80, CD86 and MHC class II) due to prominent interleukin‐10 (IL‐10) production while augmenting the expression of tolerogenic molecules including PD‐L1, CD103, Tim‐3 and indoleamine 2,3‐dioxygenase on DCs in a multiplicity‐of‐infection (MOI) ‐dependent manner. These results indicate that virulent M. tuberculosis drives immature DCs toward a tolerogenic phenotype. Notably, the tolerogenic phenotype of H37Rv‐infected DCs was blocked in DCs generated from IL‐10^−/− mice or DCs treated with an IL‐10‐neutralizing monoclonal antibody, leading to restoration of Th1 polarization. These findings suggest that IL‐10 induces a tolerogenic DC phenotype. Interestingly, p38 mitogen‐activated protein kinase (MAPK) activation predominantly mediates IL‐10 production; hence, H37Rv tends to induce a tolerogenic DC phenotype through expression of tolerogenic molecules in the p38 MAPK–IL‐10 axis. Therefore, suppressing the tolerogenic cascade in DCs is a novel strategy for stimulating optimal protective T‐cell responses against M. tuberculosis infection.     

Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells

Leptin is an adipose‐secreted hormone that plays an important role in both metabolism and immunity. Leptin has been shown to induce Th1‐cell polarization and inhibit Th2‐cell responses. Additionally, leptin induces Th17‐cell responses, inhibits regulatory T (Treg) cells and modulates autoimmune diseases. Here, we investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and Treg‐cell immune responses in mice. We observed that leptin deficiency (i) reduced the expression of DC maturation markers, (ii) decreased DC production of IL‐12, TNF‐α, and IL‐6, (iii) increased DC production of TGF‐β, and (iv) limited the capacity of DCs to induce syngeneic CD4⁺ T‐cell proliferation. As a consequence of this unique phenotype, DCs generated under leptin‐free conditions induced Treg or T_H17 cells more efficiently than DCs generated in the presence of leptin. These data indicate important roles for leptin in DC homeostasis and the initiation and maintenance of inflammatory and regulatory immune responses by DCs.     

Regulation of the tolerogenic function of steady‐state DCs

Dendritic cells (DCs) are master regulators of T‐cell responses. After sensing pathogen‐derived molecular patterns (PAMPs), or signals of inflammation and cellular stress, DCs differentiate into potent activators of naïve CD4⁺ and CD8⁺ T cells through a process that is termed DC maturation. By contrast, DCs induce and maintain peripheral T‐cell tolerance in the steady state, that is in the absence of overt infection or inflammation. However, the immunological steady state is not devoid of DC‐activating stimuli, such as commensal microorganisms, subclinical infections, or basal levels of proinflammatory mediators. In the presence of these activating stimuli, DC maturation must be calibrated to ensure self‐tolerance yet allow for adequate T‐cell responses to infections. Here, we review the factors that are known to control DC maturation in the steady state and discuss their effect on the tolerogenic function of steady‐state DCs.     

Streptococcus mutans Wall‐Associated Protein A Promotes TLR4‐Induced Dendritic Cell Maturation

Dendritic cells orchestrate innate and adaptive immune responses, which are central to establishing efficient responses to vaccination. Wall‐associated protein A (WapA) of Streptococcus mutans was previously used as a vaccine in animal studies for immunization against dental caries. However, as a cell surface protein, whether WapA activates innate immune responses and the effects of WapA on DCs remain unclear. In this study, WapA was cloned into the GST fusion vector pEBG, which can be expressed efficiently in mammalian cells. We found that when added before stimulation with LPS, purified WapA‐GST protein increased TLR4‐induced NF‐κB and MAPK signalling pathway activation. Pretreatment with WapA‐GST also increased LPS‐induced proinflammatory cytokine production by DCs, including IL‐12, IL‐6 and TNF‐α. Furthermore, expression of the DC maturation markers CD80/86, CD40 and MHC II was also increased by WapA pretreatment. These data indicate that WapA is recognized by DCs and promotes DC maturation.     

Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5′‐triphosphate‐binding cassette transporters far beyond an efflux pump

This study examines adenosine 5′‐triphosphate‐binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC‐transporter inhibitors. Monocyte‐derived DCs were cultured in the presence of interleukin (IL)‐4/granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi‐drug resistance (MDR1) and multi‐drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P‐glycoprotein (Pgp) expression with confocal microscopy. Up‐regulation of maturation markers was observed in hypoxia and LPS‐DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional‐like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia‐DCs induced more B lymphocyte proliferation than control‐iDC (56% versus 9%), while LPS‐DCs induced more CD8‐lymphocyte proliferation (67% versus 16%). ABC transporter‐inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC₅₀): P‐glycoprotein inhibition using valspodar (PSC833) 5 μM, CAS 115104‐28‐4 (MK571) 50 μM and probenecid 2·5 μM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated‐DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC‐transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia.     

Dendritic cell functions in the inductive and effector sites of intestinal immunity

The intestine is constantly exposed to foreign antigens, which are mostly innocuous but can sometimes be harmful. Therefore, the intestinal immune system has the delicate task of maintaining immune tolerance to harmless food antigens while inducing tailored immune responses to pathogens and regulating but tolerating the microbiota. Intestinal dendritic cells (DCs) play a central role in these functions as sentinel cells able to prime and polarize the T cell responses. DCs are deployed throughout the intestinal mucosa but with local specializations along the gut length and between the diffuse effector sites of the gut lamina propria (LP) and the well-organized immune inductive sites comprising isolated lymphoid follicles (ILFs), Peyer's patches (PPs), and other species-specific gut-associated lymphoid tissues (GALTs). Understanding the specificities of each intestinal DC subset, how environmental factors influence DC functions, and how these can be modulated is key to harnessing the therapeutic potential of mucosal adaptive immune responses, whether by enhancing the efficacy of mucosal vaccines or by increasing tolerogenic responses in inflammatory disorders. In this review, we summarize recent findings related to intestinal DCs in steady state and upon inflammation, with a special focus on their functional specializations, highly dependent on their microenvironment.     

DC‐SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis

Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC‐SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC‐SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC‐SIGN was co‐expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC‐SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti‐DC‐SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC‐SIGN, major histocompatibility complex (MHC) class II and CD80 was up‐regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)‐γ/interleukin (IL)‐4 ratio increased. However, anti‐DC‐SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC‐like function through their expression of DC‐SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC‐SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects.