HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.     

Newly activated T cells promote maturation of bystander dendritic cells but not IL-12 production

The activation of dendritic cells (DC) leads to increased costimulatory activity (termed DC maturation) and, in some instances, production of immunomodulatory cytokines such as IL-12. Both innate and T cell-derived signals can promote DC activation but it is unclear to what extent the two classes of stimuli are interchangeable or regulate distinct aspects of DC function. In this study, we show that signals from newly activated CD4(+) T cells cannot initiate IL-12 synthesis although they can amplify secretion of bioactive IL-12 p70 by DC exposed to an appropriate innate stimulus. This occurs exclusively in cis and does not influence IL-12 synthesis by bystander DC that do not present Ag. In marked contrast, signals from newly activated CD4(+) T cells can induce an increase in DC costimulatory activity in the absence of any innate priming. This occurs both in cis and in trans, affecting all DC in the microenvironment, including those that do not bear specific Ag. Consistent with the latter, we show that newly activated CD4(+) T cells in vivo can deliver "help" in trans, effectively lowering the number of MHC/peptide complexes required for proliferation of third-party naive CD4(+) T cells recognizing Ag on bystander DC. These results demonstrate that DC maturation and cytokine production are regulated distinctly by innate stimuli vs signals from CD4(+) T cells and reveal a process of trans activation of DC without secretion of polarizing cytokines that takes place during T cell priming and may be involved in amplifying immune responses.     

Rapid downregulation of antigen processing enzymes in ex vivo generated human monocyte derived dendritic cells occur endogenously in extended cultures

Dendritic cells, the most potent antigen presenting cells, have been shown in murine models to induce immune responses against many antigens. Their role in the initiation of antitumour immunity has received enormous attention. Their ability to process and present antigen is dependent on their state of maturation. This study examines the activity of human monocyte-derived dendritic cells at two different time points and the corresponding changes in the proteolytic enzyme activity. Dendritic cells were produced from peripheral blood mononuclear cells of normal volunteers. Plastic adherent cells were cultured for 5 or 7 days with recombinant human (rh)GM-CSF and rhIL-4. Flow cytometry showed that day 5 dendritic cells (DC) were less mature than day 7 DC as indicated by the expression of CD1a, CD11c, CD14, CD80, CD83, CD86 and MHC-II. Proteolytic activity of the enzymes cathepsin C and cathepsin G and phagocytosis of particulate antigens also showed significant differences between d5 dendritic cells and d7 dendritic cells. Allogeneic costimulatory activity of d7 dendritic cells was also significantly increased. Induction of immunity requires active presentation of antigens by antigen processing cells on their MHC-I and/or MHC-II molecules. Study of peptide carriers and peptide precursor molecules showed a significant decrease in CLIP levels in the day 7 DC, suggesting their decreased ability to process antigens but no difference in their ability to load MHC-II molecules. These findings indicate that the length of time in culture, in the absence of exogenous maturation - inducing stimuli affects dendritic cell maturation. Intracellular enzymatic activities of dendritic cells also changed rapidly with small changes in phenotype.     

Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.     

Antigen-processing machinery in human dendritic cells: up-regulation by maturation and down-regulation by tumor cells

It has been known for some time that functional properties of dendritic cells (DC), and in particular their ability to process and present Ags to T cells, can be modulated by cytokine-induced maturation and by interactions with tumor cells. However, the molecular basis for these functional changes is unknown. We have investigated whether changes in expression of Ag-processing machinery (APM) components in DC are associated with alterations in their ability to present tumor-derived Ags to T cells. Using a panel of mAbs specific for individual APM components and a quantitative flow cytometry-based method, the level of APM components was measured in DC generated from peripheral blood monocytes of 12 normal donors and of 8 patients with cancer. Immature DC had significantly lower (p < 0.01) expression of MB1, LMP-7, LMP-10, TAP-1, and tapasin than mature DC. However, maturation in the presence of a cytokine mixture up-regulated expression of these components in DC obtained from normal donors and patients with cancer. Immature DC incubated with tumor cells had significantly lower (p < 0.001) expression of MB1, LMP-2, LMP-7, LMP-10, and endoplasmic reticulum p75 than controls. These changes were associated with a decreased ability of DC to present tumor-derived Ags to T cells, as measured in ELISPOT assays and with apoptosis of T cells in DC-T cell cultures. Thus, tumor cells have a significant suppressive effect on DC; however, ex vivo maturation of DC derived from patients with cancer in a polarizing cytokine mix restores normal expression of APM components and Ag-processing capabilities.     

MyD88-dependent TLR1/2 signals educate dendritic cells with gut-specific imprinting properties

Gut-associated dendritic cells (DC) synthesize all-trans retinoic acid, which is required for inducing gut-tropic lymphocytes. Gut-associated DC from MyD88(-/-) mice, which lack most TLR signals, expressed low levels of retinal dehydrogenases (critical enzymes for all-trans retinoic acid biosynthesis) and were significantly impaired in their ability to induce gut-homing T cells. Pretreatment of extraintestinal DC with a TLR1/2 agonist was sufficient to induce retinal dehydrogenases and to confer these DC with the capacity to induce gut-homing lymphocytes via a mechanism dependent on MyD88 and JNK/MAPK. Moreover, gut-associated DC from TLR2(-/-) mice, or from mice in which JNK was pharmacologically blocked, were impaired in their education to imprint gut-homing T cells, which correlated with a decreased induction of gut-tropic T cells in TLR2(-/-) mice upon immunization. Thus, MyD88-dependent TLR2 signals are necessary and sufficient to educate DC with gut-specific imprinting properties and contribute in vivo to the generation of gut-tropic T cells.     

Dendritic cells: making progress with tumour regression?

Due to their potent ability to activate the immune system, dendritic cells (DC) are showing promise as potential adjuvants for tumour immunotherapy of cancer patients. However, little is known about the effect tumour cells can have on DC function. Indeed, the discovery of different DC subsets with different immunological functions indicates that the relationship between tumour cells and tumour-infiltrating DC subtypes is likely to be complex. There remains a lot to be understood about the effects of tumours on DC before we can expect to benefit from DC-based tumour immunotherapy of cancer patients. Here we review the recent advances being made in understanding DC phenotype and function in relation to interactions with different types of tumours.     

Characterization of the biological activities of uridine diphosphate in human dendritic cells: Influence on chemotaxis and CXCL8 release

Uridine nucleotides are endogenous nucleotides which are released into the extracellular space from mechanical stressed endothelial and epithelial cells as well as lipopolysaccharide (lps)-stimulated monocytes. Here, we studied the biological activity of the selective purinoreceptor P2Y6 (P2YR6) agonist Uridine 5'diphosphate (UDP) as well as the P2YR2- and P2YR4-activating uridine 5'triphosphate (UTP) on human dendritic cells (DC). These cells in their immature state have the ability to migrate from blood to peripheral target sites where they sense dangerous signals and capture potential antigens. Moreover, mature DC induce innate immune responses and migrate from peripheral tissues to secondary lymphoid organs in order to activate naive T cells and initiate adaptive immunity. Here, we were able to show that uridine nucleotides stimulated Ca(2+) transients, actin polymerization, and chemotaxis in immature DC. Experiments with pertussis toxin, the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDPgammaS) and receptor antagonists, as well as desensitization studies suggested that these uridine nucleotides activities were mediated by different G(i) protein-coupled receptors. During lps-induced maturation, DC lost their ability to respond towards uridine nucleotides with these activities. Instead, UDP, but not UTP, stimulated the release of the CXC-chemokine 8 (CXCL8) from mature DC in a reactive blue sensitive manner. Moreover, our study indicates that UDP stimulates different signaling pathways in immature and mature DC in order to favor the accumulation of immature DC and to augment the capacity to secrete CXCL8 in mature DC.     

The human thymic dendritic cell phenotype and its modification in culture.

In order to extend our study of human thymic dendritic cells (DC) we have purified DC by density gradient separation followed by treatment with CD1 and CD2 mAb and antibody-coated immunobeads. The resulting population contains 60 to 75% brightly HLA-DR+ cells. Morphological and functional studies demonstrate that these cells share the common characteristics of dendritic cells. Extensive phenotypic analysis of the purified DC has been made using a panel of mAb. Cytofluorometric assays with mAb reactive with common leucocyte antigen confirm that the brightly HLA-DR+ cells are of mesenchymal origin. Thymic DC express HLA-DQ and HLA-class I antigens. They are also positive for the expression of CD45RA molecules and some express the ICAM-1 and the LFA-1 molecules. DC do not stain with a wide variety of anti-T, -B, and -monocyte or -M phi mAb and lack Fc gamma RIII, CR2, and CR3. Freshly isolated DC failed to stain with OKT6 mAb; however, they progressively acquire the CD1 molecule after a few days culture. The acquisition of CD1 molecule is selective since CD4, CD2, and HLA-ABC molecules are not upregulated under the same conditions. From phenotypic results, it was therefore possible to sort brightly HLA-DR+ or -DQ+ cells and so obtain greater than 90 to 95% purified human thymic DC. Such homogeneous DC populations are obviously of great interest for the study of thymic DC functions.     

NK cell TRAIL eliminates immature dendritic cells in vivo and limits dendritic cell vaccination efficacy

Recent studies have implicated a possible role for NK cells in regulating dendritic cells (DC) in vitro. In the present study, we demonstrate that immature DC are rapidly eliminated by NK cells in vivo via a pathway dependent on the TNF-related apoptosis-inducing ligand (TRAIL). Elimination of NK cells and/or neutralization of TRAIL function during immunization with immature DC loaded with nonself or tumor Ags significantly enhanced T cell responses to these Ags and Ag-specific tumor immunity. These data suggested that NK cell TRAIL might regulate responses to vaccination by controlling the survival of Ag-loaded DC.     

Natural killer dendritic cells have both antigen presenting and lytic function and in response to CpG produce IFN-gamma via autocrine IL-12

We have isolated rare cells bearing the NK cell surface marker NK1.1, as well as the dendritic cell (DC) marker CD11c, from the spleen, liver, lymph nodes, and thymus of normal mice. These cells possess both NK cell and DC function because they can lyse tumor cells and subsequently present Ags to naive Ag-specific T cells. Interestingly, in response to IL-4 plus either IL-2 or CpG, NKDC produce more IFN-gamma than do DC, or even NK cells. We determined that CpG, but not IL-2, induces NKDC to secrete IFN-gamma via the autocrine effects of IL-12. In vivo, CpG dramatically increases the number of NKDC. Furthermore, NKDC induce greater Ag-specific T cell activation than do DC after adoptive transfer. Their unique ability to lyse tumor cells, present Ags, and secrete inflammatory cytokines suggests that NKDC may play a crucial role in linking innate and adaptive immunity.     

Phagocytosis induces lysosome remodeling and regulated presentation of particulate antigens by activated dendritic cells

Immunization with particulate Ag effectively induces antitumor and antiviral T cell-mediated immunity. Immature dendritic cells (DCs) efficiently internalize, process, and present a variety of particulate Ags; however, previously published data suggest that both the uptake of soluble Ag through micropinocytosis, and phagocytosis of particulates are significantly curtailed in activated DC populations. In this study, we demonstrate that although macropinocytosis of soluble Ag is diminished following DC activation, subsets of DCs in activated DC populations retain the ability to actively phagocytose particulate Ags. Live cell imaging of activated DCs reveals that phagocytosis of particulates can result in cytoskeletal remodeling and perinuclear lysosome cluster disruption in a time-dependent manner. Interestingly, our results suggest that in activated DC populations, presentation of phagocytosed particulate Ags is dependent on the nature of the activation signal. These results provide direct evidence of functional heterogeneity in DC populations and contribute to the development of particle-based immunization strategies.     

Angiotensin-converting enzyme limits inflammation elicited by Trypanosoma cruzi cysteine proteases: a peripheral mechanism regulating adaptive immunity via the innate kinin pathway

Tissue injury by pathogens induces a stereotyped inflammatory response that alerts the innate immune system of the potential threat to host integrity. Here, we review knowledge emerging from investigations of the role of the kinin system in the mechanisms that link innate to the adaptive phase of immunity. Progress in this field started with results demonstrating that bradykinin is an endogenous danger signal that induces dendritic cell (DC) maturation via G protein-coupled bradykinin B2 receptors (B2R). The immunostimulatory role of kinins was recently confirmed in two different mouse models of Trypanosoma cruzi infection, a parasitic protozoan equipped with kinin-releasing cysteine proteases (cruzipain). Infection by the intraperitoneal route showed that DCs from B2R-/- mice (susceptible phenotype) failed to sense kinin 'danger' signals proteolytically released by parasites, explaining why these mutant mice display lower frequencies of interferon-gamma-producing effector T-cells. Studies of the dynamics of inflammation in the subcutaneous model of infection revealed that the balance between cruzipain and angiotensin-converting enzyme, respectively acting as kinin-generating and degrading enzymes, governs extent of DC maturation and TH1 development via the B2R-dependent innate pathway. Studies of the kinin role in immunity may shed light on the relationship between proteolytic networks and the cytokine circuits that guide T-cell development.     

Dendritic cell programming by cytomegalovirus stunts naive T cell responses via the PD-L1/PD-1 pathway

Early during infection, CMV targets dendritic cells (DC) and alters their functions. Herein we show that CMV-infected DC maintain the ability to present both virus-derived and exogenous Ags, but that they actively induce tolerance or anergy in Ag-specific T cells. CMV accomplishes this by selectively maintaining high-level expression of the negative costimulatory molecule programmed death ligand-1 (PD-L1), while commensurately down-regulating positive costimulatory molecules and MHC on the DC surface. Consequently, CD4 and CD8 T cells activated by these infected DC have a stunted phenotype, characterized by poor proliferation, effector function. and recall responses. Blocking PD-L1, but not PD-L2, during direct priming of naive T cells by infected DC significantly restores Ag-specific T cell functions. Using systems where direct and cross-priming of T cells can be distinguished revealed that PD-L1/PD-1 signaling contributes only when naive T cells are primed directly by infected DC, and not upon cross-presentation of viral Ags by uninfected DC. These data suggest that murine CMV programs infected DC during acute infection to inhibit early host adaptive antiviral responses by tipping the balance between negative and positive cosignals.     

Targeting dendritic cells for priming cellular immune responses

The cardinal role of dendritic cells (DC) in priming adaptive immunity and in orchestrating immune responses against all classes of pathogens and also against tumors is well established. Their unique potential both to maintain self-tolerance and to initiate protective immune responses against foreign and/or dangerous structures is based on the functional diversity and flexibility of these cells. Tissue DC lining antigenic portals such as mucosal surfaces and the skin are specialized to take up a wide array of compounds including proteins, lipids, carbohydrates, glycoproteins, glycolipids and oligonucleotides, particles carrying such structures and apoptotic or necrotic cells. This process is facilitated by specialized receptors with high endocytic capacity, which provides potential targets for delivering designed molecules. The best route for targeting B- and/or T cell epitopes, however, is still the subject of intense investigation. Immature DC, which reside in various tissues, can be activated by pathogens, stress and inflammation or modified metabolic products, which induce mobilization of cells to draining lymph nodes where they act as highly potent professional antigen presenting cells. This is brought about by the ability to present their accumulated intracellular content for both CD4+ helper (Th) and CD8+ cytotoxic/cytolytic T lymphocytes (Tc/CTL). Engulfed proteins are processed intracellularly and their peptide fragments are transported to the cell surface in the context of major histocompatibility complex encoded class I and II molecules for presentation to Th cells and CTLs, respectively. The T cell priming capacity of DC, however, depends not only on antigen presentation but also on other features of DC. Human monocyte-derived DC provide an excellent tool to study the internalizing, antigen-presenting and T cell-activating functions of DC at their immature and activated differentiation states. These biological activities of DC, however, are highly dependent on their migratory potential from the peripheral non-lymphoid tissues to the lymph nodes, on the expression of adhesion molecules, which support the interaction of DC with T lymphocytes, and the cytokines secreted by DC, which polarize immune responses to Th1-mediated cellular or Th2-mediated antibody responses. These results altogether demonstrate that monocyte-derived DC are useful candidates for in vitro or in vivo targeting of antigens to induce efficient adaptive immune responses against pathogens and also against tumors.     

Modified lipoproteins provide lipids that modulate dendritic cell immune function

Both physiological and pathological situations can result in biochemical changes of low-density lipoproteins (LDL). Because they can deliver signals to dendritic cells (DC), these modified lipoproteins now appear as regulators of the immune response. Among these modified lipoproteins, oxidized LDL (oxLDL) that accumulate during inflammatory conditions have been extensively studied. Numerous studies have shown that oxLDL induce the maturation of DC, enhancing their ability to activate IFNγ secretion by T cells. LDL treated by secreted phospholipase A₂ also promote DC maturation. Among the bioactive lipids generated by oxidation or phospholipase treatment of LDL, lysophosphatidylcholine (LPC) and some saturated fatty acids induce DC maturation whereas some unsaturated fatty acids or oxidized derivatives have opposite effects. Among other factors, the nuclear receptor peroxisome-proliferator activated receptor γ (PPARγ) plays a crucial role in this regulation. Non-modified lipoproteins also contribute to the regulation of DC function, suggesting that the balance between native and modified lipoproteins, as well as the biochemical nature of the LDL modifications, can regulate the activation threshold of DC. Here we discuss two pathological situations in which the impact of LDL modifications on inflammation and immunity could play an important role. During atherosclerosis, modified LDL accumulating in the arterial intima may interfere with DC maturation and function, promoting a Th1 immune response and a local inflammation favoring the development of the pathology. In patients chronically infected, the hepatitis C virus (HCV) interferes with lipoprotein metabolism resulting in the production of infectious modified lipoproteins. These lipo-viral-particles (LVP) are modified low-density lipoproteins containing viral material that can alter DC maturation and affect specific toll-like receptor signaling. In conclusion, lipoprotein modifications play an important role in the regulation of immunity by delivering signals of danger to DC and modulating their function.     

Human neonatal dendritic cells are competent in MHC class I antigen processing and presentation

Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E-restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens.     

Investigation into the prevalence of a novel dendritic‐like cell subset in vivo

A novel dendritic‐like cell subset termed L‐DC was recently identified in murine spleen based on marker expression of a homogeneous cell population derived from long‐term culture of neonatal spleen. The function of L‐DC is distinct from other splenic dendritic and myeloid cell subsets because of their high endocytic capacity and their ability to cross‐present antigen to CD8⁺ T cells. This paper shows the subset to be unique to spleen and blood, with a similar, but possibly functionally distinct subset also present in bone marrow. The prevalence of the subset is low; ~6% of all dendritic and myeloid cells in the spleen and ~5% in blood. However, they are a distinct cell type on the basis of marker expression, and endocytic and T‐cell stimulatory capacity. Attempts to identify an enriched population of these cells in mutant mouse strains with reported increases in myelopoiesis showed either a lack of L‐DC or an altered phenotype reflective of the phenotype of the mouse strain.     

Opposing signals from pathogen-associated molecular patterns and IL-10 are critical for optimal dendritic cell induction of in vivo humoral immunity to Streptococcus pneumoniae

Interleukin10 is widely regarded as an inhibitor of immunity in part through its ability to inhibit dendritic cell (DC) function. The present study suggests a modification of this view by demonstrating instead that a critical balance exists between signals mediated by pathogen-associated molecular patterns and IL-10 for optimization of DC induction of an in vivo humoral immune response. Bone marrow-derived, CD8alpha(-) DC pulsed with Streptococcus pneumoniae in vitro induce in vivo protein- and polysaccharide-specific Ig isotype responses upon adoptive transfer into naive mice. Following bacterial activation, DC have a limited time during which they can function as effective APCs in vivo due to the onset of maturation-associated apoptosis. Autocrine IL-10, by limiting the time during which DC are responsive to widely varying levels of bacterial stimulation, delays the onset of DC apoptosis and thus prolongs the time during which DC are able to elicit in vivo humoral immunity. These data demonstrate a requirement for properly balanced positive and negative signaling in DC to optimize an in vivo immune response to a pathogen.     

Induction of regulatory T cells and dominant tolerance by dendritic cells incapable of full activation

Transplants tolerated through a process known as infectious tolerance evoke continuous recruitment of regulatory T (Treg) cells that are necessary to maintain the unresponsive state. This state is maintained long-term and requires continuous Ag exposure. It is not known, however, whether infectious tolerance operates through sustained recruitment of pre-existing regulatory cells, induction of regulatory cells, or both. Using mice deficient in natural Treg cells, we show here that quiescent donor dendritic cells (DC) laden with histocompatibility Ag can induce Treg cells de novo that mediate transplantation tolerance. In contrast, fully activated DC fail to do so. These findings suggest that DC incapable of delivering full activation signals to naive T cells may favor their polarization toward a regulatory phenotype. Furthermore, they suggest a role for quiescent endogenous DC in the maintenance of the tolerant state.