The involvement of TNF-alpha-related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN-beta-stimulated human dendritic cells to tumor cells

TNF-alpha-related apoptosis-inducing ligand (TRAIL) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells. Dendritic cells (DCs), besides their role as APCs, now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells. Here, we report that both human CD34(+) stem cell-derived DCs (CD34DCs) and human CD14(+) monocyte-derived DCs (MoDCs) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL. Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes. The level of TRAIL expression remained constant until DC maturation. TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-beta stimulation. Moreover, IFN-beta could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells, but LPS did not have such an effect. The up-regulation of TRAIL on IFN-beta-stimulated DCs partially contributed to the increased cytotoxicity of DCS: Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-beta-stimulated DCS: In contrast, NF-kappaB inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCS: Our studies demonstrate that IFN-beta-stimulated DCs are functionally cytotoxic. Thus, an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL. Subsequently, DCs act as APCs involved in the uptake, processing, and presentation of apoptotic tumor Ags to cross-prime CD8(+) CTL cells.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Recombinant monomeric CD40 ligand for delivering polymer particles to dendritic cells

Dendritic cells (DCs) are considered the most efficient antigen-presenting cells and are therefore ideal targets for in vivo delivery of antigen for vaccines. We are investigating the strategy of using CD40 ligand (CD40L) as a targeting moiety because this protein has the potential to not only target DCs, but also stimulate cell maturation, leading to more potent immune responses. We have shown that a recombinant, monomeric CD40 ligand fusion protein conjugated to polystyrene micro- and nanoparticles led to significantly enhanced uptake by DCs in vitro. This enhancement was observed for particles of both sizes and in both a murine DC cell line and primary DCs. The uptake appeared to be specifically mediated by CD40L binding to CD40 expressed on DCs. Enhanced uptake of nanoparticles in draining lymph nodes of mice was not observed, however, 48 hours after subcutaneous injection. These findings suggest that CD40 ligand may be a potentially useful targeting moiety for delivery of particulate vaccines to DCs, and that further optimization of both CD40L and the polymer carriers is necessary to achieve efficacy in vivo.     

Consequences of Fas-mediated human dendritic cell apoptosis induced by measles virus

Mortality from measles virus (MV) infection is caused mostly by secondary infections associated with a pronounced immunosuppression. Dendritic cells (DCs) represent a major target of MV and could be involved in immunosuppression. In this study, human monocyte-derived DCs were used to demonstrate that DC apoptosis in MV-infected DC-T-cell cocultures is Fas mediated, whereas apoptotic T cells could not be rescued by blocking the Fas pathway. Two novel consequences of DC apoptosis after MV infection were demonstrated. (i) Fas-mediated apoptosis of DCs facilitates MV release, while CD40 activation enhances MV replication in DCs. Indeed, detailed studies of infectious MV release and intracellular MV nucleoprotein (NP) showed that inhibition of CD40-CD40L ligand interaction blocks NP synthesis. We conclude that the CD40 ligand expressed by activated T cells first enhances MV replication in DCs, and then Fas ligand produced by activated T cells induces Fas-mediated apoptosis of DCs, thus facilitating MV release. (ii) Not only MV-infected DCs but also bystander uninfected DCs undergo a maturation process confirmed by CD1a, CD40, CD80, CD86, CD83, and major histocompatibility complex type II labeling. The bystander maturation effect results from contact and/or engulfment of MV-induced apoptotic DCs by uninfected DCs. A model is proposed to explain how both a specific immune response and immunosuppression can simultaneously occur after MV infection through Fas-mediated apoptosis and CD40 activation of DCs.     

The duration of signaling through CD40 directs biological ability of dendritic cells to induce antitumor immunity

Although it has been demonstrated that the functions of dendritic cells (DCs), including Ag capture, Ag presentation, and migratory activity, change dynamically with their maturation, the most appropriate conditioning of DCs for anticancer immunotherapy is still unclear. The help signal is one of the most potent stimuli for DC maturation and is provided by the interaction of CD40 expressed on DCs with CD40 ligand on CD4(+) T cells. To elucidate the appropriate conditioning of DCs for anticancer immunotherapy, we examined the biological activity of DCs stimulated with immobilized anti-CD40 Ab. DCs stimulated for 3 h (3h-DCs) still showed an immature phenotype, but exhibited augmented migration toward secondary lymphoid tissues. Subcutaneous injection of 3h-DCs facilitated priming of T cells, which could mediate potent antitumor therapeutic efficacy, in draining lymph nodes and successfully induced protective immunity. In contrast, 24h-DCs showed a mature phenotype with good Ag presentation ability to induce cell killing by adoptively transferred CD8(+) T cells when injected at tumor sites; however, they showed no migratory activity and were unable to induce protective immunity when injected s.c. This is the first report that functionally distinct DCs, either for the priming phase or for the effector phase, could be obtained by conditioning with CD40 stimulation and that the duration of stimulation determines the biological outcome. The usage of DCs conditioned for the priming phase might provide significant advantages in anticancer immunotherapy.     

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1beta, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.     

HIV induces maturation of monocyte-derived dendritic cells and Langerhans cells

In HIV infection, dendritic cells (DCs) may play multiple roles, probably including initial HIV uptake in the anogenital mucosa, transport to lymph nodes, and subsequent transfer to T cells. The effects of HIV-1 on DC maturation are controversial, with several recent conflicting reports in the literature. In this study, microarray studies, confirmed by real-time PCR, demonstrated that the genes encoding DC surface maturation markers were among the most differentially expressed in monocyte-derived dendritic cells (MDDCs), derived from human blood, treated with live or aldrithriol-2-inactivated HIV-1(BaL). These effects translated to enhanced cell surface expression of these proteins but differential expression of maturation markers was only partial compared with the effects of a conventional potent maturation stimulus. Such partially mature MDDCs can be converted to fully mature cells by this same potent stimulus. Furthermore, live HIV-1 stimulated greater changes in maturation marker surface expression than aldrithriol-2-inactivated HIV-1 and this enhanced stimulation by live HIV-1 was mediated via CCR5, thus suggesting both viral replication-dependent and -independent mechanisms. These partially mature MDDCs demonstrated enhanced CCR7-mediated migration and are also able to stimulate interacting T cells in a MLR, suggesting DCs harboring HIV-1 might prepare CD4 lymphocytes for transfer of HIV-1. Increased maturation marker surface expression was also demonstrated in native DCs, ex vivo Langerhans cells derived from human skin. Thus, HIV initiates maturation of DCs which could facilitate subsequent enhanced transfer to T cells.     

Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-alpha or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1alpha/beta and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.     

Increased extracellular pressure provides a novel adjuvant stimulus for enhancement of conventional dendritic cell maturation strategies

Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40 mmHg increased pressure for 12 h, then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.     

Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram-positive bacteria to CD4+ T lymphocytes.

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.     

The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells

Toll-like receptors (TLRs) are pattern recognition receptors that serve an important function in detecting pathogens and initiating inflammatory responses. Upon encounter with foreign Ag, dendritic cells (DCs) go through a maturation process characterized by an increase in surface expression of MHC class II and costimulatory molecules, which leads to initiation of an effective immune response in naive T cells. The innate immune response to bacterial flagellin is mediated by TLR5, which is expressed on human DCs. Therefore, we sought to investigate whether flagellin could induce DC maturation. Immature DCs were cultured in the absence or presence of flagellin and monitored for expression of cell surface maturation markers. Stimulation with flagellin induced increased surface expression of CD83, CD80, CD86, MHC class II, and the lymph node-homing chemokine receptor CCR7. Flagellin stimulated the expression of chemokines active on neutrophils (IL-8/CXC chemokine ligand (CXCL)8, GRO-alpha/CXCL1, GRO-beta/CXCL2, GRO-gamma/CXCL3), monocytes (monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2), and immature DCs (macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4), but not chemokines active on effector T cells (IFN-inducible protein-10 kDa/CXCL10, monokine induced by IFN-gamma/CXCL9, IFN-inducible T cell alpha chemoattractant/CXCL11). However, stimulating DCs with both flagellin and IFN-inducible protein-10 kDa, monokine induced by IFN-gamma, and IFN-inducible T cell alpha chemoattractant expression, whereas stimulation with IFN-beta or flagellin alone failed to induce these chemokines. In functional assays, flagellin-matured DCs displayed enhanced T cell stimulatory activity with a concomitant decrease in endocytic activity. Finally, DCs isolated from mouse spleens or bone marrows were shown to not express TLR5 and were not responsive to flagellin stimulation. These results demonstrate that flagellin can directly stimulate human but not murine DC maturation, providing an additional mechanism by which motile bacteria can initiate an acquired immune response.     

Activated platelets rapidly up-regulate CD40L expression and can effectively mature and activate autologous ex vivo differentiated DC

Background DC are a promising immunotherapeutic for treatment of infectious/malignant disease. For clinical trials, immature DC generated from precursor cells such as monocytes, using serum-free media containing GM-CSF and IL-4, can be matured with specific cytokines/factors. CD40 ligand (CD40L) plays an important role in DC activation/maturation but is not available for clinical applications. These studies evaluated the feasibility of using activated platelets with elevated CD40L surface expression to stimulate autologous DC maturation. Methods Pilot and clinical scale studies were executed using magnetic/centrifugal separation. Monocyte precursors were differentiated to immature DC with GM-CSF and IL-4 and the ability of activated autologous platelets to mature these cells was evaluated on the basis of phenotype and function. Results In small-scale studies, DC cultures stimulated with activated autologous platelets (CD40L-AP), tumor necrosis factor-alpha (TNF-alpha) or soluble CD40L (sCD40L) up-regulated expression of phenotype markers indicative of activation and maturation. CD86 expression was significantly enhanced (P<0.05) by stimulation with either CD40L-AP (75.5+/-14.5%) or sCD40L (80.5%+/-5.3%) compared with immature DC (55.2+/-14.8%), as were CD80 and CD83. Similarly, in large-scale studies using Isolex 300I to enrich for monocytes and platelets for DC generation/maturation on a clinical scale, stimulation with CD40L-AP increased CD86 and CD80 expression as well as the ability to stimulate allogeneic lymphocytes compared with control cultures. Discussion These results demonstrate that thrombin-activated platelets express CD40L and are effective at inducing matured DC with potent immunogenic activity. Furthermore, these studies demonstrate the feasibility of this approach for clinical immunotherapeutic interventions.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

CD40 ligand enhances dengue viral infection of dendritic cells: a possible mechanism for T cell-mediated immunopathology

We have previously shown that dengue virus (DV) productively infects immature human dendritic cells (DCs) through binding to cell surface DC-specific ICAM-3-grabbing nonintegrin molecules. Infected DCs are apoptotic, refractory to TNF-alpha stimulation, inhibited from undergoing maturation, and unable to stimulate T cells. In this study, we show that maturation of infected DCs could be restored by a strong stimulus, CD40L. Addition of CD40L significantly reduced apoptosis of DCs, promoted IL-12 production, and greatly elevated the IFN-gamma response of T cells, but yet did not restore T cell proliferation in MLR. Increased viral infection of DCs was also observed; however, increased infection did not appear to be mediated by DC-specific ICAM-3-grabbing nonintegrin, but rather was regulated by decreased production of IFN-alpha and decreased apoptotic death of infected DCs. Because CD40L is highly expressed on activated memory (but not naive) T cells, the observation that CD40L signaling results in enhanced DV infection of DC suggests a possible T cell-dependent mechanism for the immune-mediated enhancement of disease severity associated with some secondary dengue infections.     

Quantities of interleukin-12p40 in mature CD8alpha negative dendritic cells correlate with strength of TCR signal and determine Th cell development

Strength of T cell antigen receptor (TCR) signaling drives the development of Th1 and Th2 subsets from naive T helper precursors. The quantity of interleukin-12 (IL-12) from antigen presenting cells (APC) is also profoundly involved in Th development. TCR signal strength and IL-12 production from dendritic cells (DCs) are linked by CD40 ligand (CD40L) expression on activated T cells. CD40L on the activated T cells interacts with CD40 on DC, resulting in induction of IL-12 from DCs. However, the subsets of DC in spleen that produce the IL-12 have not been clearly identified. Purification of DC subsets itself may provide maturation signals to immature DCs. Thus, we used non-purified mouse spleen cells to analyze IL-12 producing cells, near to steady states, during the interaction of naive T cells either with or without agonist. Mature CD86highCD8alpha- DCs in spleen mainly produced IL-12p40 after stimulation of high dose agonist. The ratio of CD40L positive T cells and IL-12p40 secreting CD86high DCs correlated with the concentration of agonist and Th1 development. However, anti-IL-12 did not completely inhibit the Th1 development. Altogether, strength of TCR signaling directs Th cell development by regulating CD40L expression on T cells which determines production of IL-12p40 from CD86high CD8alpha- DC via CD40.     

The effect of LIGHT in inducing maturation of monocyte-derived dendritic cells from MDS patients

LIGHT is a recently cloned novel cytokine belonging to the TNF family that is selectively expressed on immature dendritic cells (iDCs) generated from monocytes isolated from human PBMCs. In these studies, we demonstrate that exogenous soluble LIGHT or soluble CD40 ligand (CD40L) can promote monocyte-derived dendritic cell maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression. Immature dendritic cells differentiated from monocytes of MDS patients displayed lower levels of costimulatory and HLA-DR molecules compared with iDCs differentiated from monocytes of normal subjects. However, upon induction of maturation by LIGHT or CD40L, the expression of costimulatory and HLA-DR molecules is comparable between DCs from MDS and normal subjects. Exogenous LIGHT- and CD40L-stimulated mature DCs (mDCs) also displayed increased antigen presentation to autologous T lymphocytes (tetanus toxin) or allogeneic T lymphocytes in mixed lymphocyte reactions. DCs matured by LIGHT showed increased secretion of IL-6, IL-12p75, and TNF-, but not IL-1. We conclude that both LIGHT and CD40L are immunoregulating factors that induce monocyte-derived iDCs from MDS patients to undergo maturation resulting in increased antigen presentation and T-cell activation. Monocyte-derived DCs can be stimulated to undergo phenotypic and functional changes with LIGHT that might be applied in the development of a DC-based vaccine for MDS treatment.