Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells.

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.     

Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway

Dendritic cells (DCs) mediate cross-priming of tumor-specific T cells by acquiring tumor Ags from dead cancer cells. The process of cross-priming would be most economical and efficient if DCs also induce death of cancer cells. In this study, we demonstrate that normal human in vitro generated immature DCs consistently and efficiently induce apoptosis in cancer cell lines, freshly isolated noncultured cancer cells, and normal proliferating endothelial cells, but not in most normal cells. In addition, in vivo generated noncultured peripheral blood immature DCs mediate similar tumoricidal activity as their in vitro counterpart, indicating that this DC activity might be biologically relevant. In contrast to immature DCs, freshly isolated monocytes (myeloid DC precursors) and in vitro generated mature DCs are not cytotoxic or are less cytotoxic, respectively, suggesting that DC-mediated killing of cancer cells is developmentally regulated. Comparable cytotoxic activity is mediated by untreated DCs, paraformaldehyde-fixed DCs, and soluble products of DCs, and is destructible by proteases, indicating that both cell membrane-bound and secreted proteins mediate this DC function. Overall, our data demonstrate that human immature DCs are capable of inducing apoptosis in cancer cells and thus to both directly mediate anticancer activity and initiate processing of cellular tumor Ags.     

Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.     

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.     

Characterization of chicken TNFR superfamily decoy receptors,DcR3 and osteoprotegerin

Tumor necrosis factor (TNF) family ligands bind to death domain-containing TNF receptors (death receptors), which can subsequently activate intracellular signaling pathways to initiate caspase activity and apoptotic cell death. Decoy receptors, without intracellular death domains, have been reported to prevent cytotoxic effects by binding to and sequestering such ligands, or by interfering with death receptor trimerization. The chicken death receptors, Fas, TNFR1, DR6, and TVB, are constitutively expressed in a relatively wide variety of hen tissues. In this study, two chicken receptors with sequence homology to the mammalian decoys, DcR3 and osteoprotegerin, were identified and their pattern of expression was characterized. Unlike the previously identified chicken death receptors, the newly characterized decoy receptors show comparatively limited expression among tissues, suggesting a tissue-specific function. Finally, characterization of these chicken receptors further contributes to understanding the evolutionary divergence of TNFR superfamily members among vertebrate species.     

Langerhans cells and dendritic cells are cytotoxic towards HPV16 E6 and E7 expressing target cells

Dendritic cells (DC) can be cytotoxic towards tumor cells by means of TNF family molecules expressed on the cell surface of activated DCs. Tumor cells expressing appropriate receptors are killed by DC, generating a source of antigen to be presented to the immune system. It has not been investigated whether Langerhans cells (LC) are selectively cytotoxic to tumor cells. This is of particular interest for epithelial tumor cells that physically interact with LC in vivo. Among epithelial tumors, the oncogenic process of cervical tumors is relatively well defined by their Human Papillomavirus (HPV) mediated etiology. To study whether HPV16 E6 and E7 expressions, otherwise observed in cervical tumor cells, can sensitize normal cervical epithelial cells to DC and LC mediated killing, the E6 and E7 genes were introduced by retroviral transfection, and cells were subsequently used as targets in cytotoxicity assays. Expression of cytotoxic molecules by effector cells was measured in response to the pro-inflammatory cytokine IFN-γ; cytotoxicity was established and concomitant expression of receptor molecules was assessed on target cells. A correlation between the shrinkage of HPV16 E6 and E7+ tumors versus DC and LC infiltration was evaluated in a murine model of cervical cancer. DC and LC proved to be equally cytotoxic towards E6 and E7 expressing cervical epithelial cells. IFN-γ induced TRAIL expression by DC and LC, and inhibition of TRAIL partially blocked cytotoxic effects. Expression of TRAIL decoy receptors was reduced following introduction of E6 and E7 into host cells. Shrinkage of HPV16 E6 and E7 expressing tumors correlated with infiltration by S100+ DC and LC, co-localizing with apoptotic mouse tumor cells. In conclusion, DC and LC mediated killing may be exploitable for anti-tumor treatment. I. Caroline Le Poole and W.M. ElMasri have contributed equally to this paper.     

Cytotoxic dendritic cells generated from cancer patients

Known for years as professional APCs, dendritic cells (DCs) are also endowed with tumoricidal activity. This dual role of DC as killers and messengers may have important implications for tumor immunotherapy. However, the tumoricidal activity of DCs has mainly been investigated in animal models. Cancer cells inhibit antitumor immune responses using numerous mechanisms, including the induction of immunosuppressive/ tolerogenic DCs that have lost their ability to present Ags in an immunogenic manner. In this study, we evaluated the possibility of generating tumor killer DCs from patients with advanced-stage cancers. We demonstrate that human monocyte-derived DCs are endowed with significant cytotoxic activity against tumor cells following activation with LPS. The mechanism of DC-mediated tumor cell killing primarily involves peroxynitrites. This observed cytotoxic activity is restricted to immature DCs. Additionally, after killing, these cytotoxic DCs are able to activate tumor Ag-specific T cells. These observations may open important new perspectives for the use of autologous cytotoxic DCs in cancer immunotherapy strategies.     

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.     

Fas and Fas ligand in gut and liver

Apoptosis (programmed cell death) has been shown to play a major role in development and in the pathogenesis of numerous diseases. A principal mechanism of apoptosis is molecular interaction between surface molecules known as the "death receptors" and their ligands. Perhaps the best-studied death receptor and ligand system is the Fas/Fas ligand (FasL) system, in which FasL, a member of the tumor necrosis factor (TNF) family of death-inducing ligands, signals death through the death receptor Fas, thereby resulting in the apoptotic death of the cell. Numerous cells in the liver and gastrointestinal tract have been shown to express Fas/FasL, and there is a growing body of evidence that the Fas/FasL system plays a major role in the pathogenesis of many liver and gastrointestinal diseases, such as inflammatory bowel disease, graft vs. host disease, and hepatitis. Here we review the Fas/FasL system and the evidence that it is involved in the pathogenesis of liver and gastrointestinal diseases.     

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G₂/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27^Kip1. In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.     

DNA fragmentation and cell death is selectively triggered in activated human lymphocytes by Fas antigen engagement.

Fas is a mouse monoclonal antibody-defined cell surface antigen of an unknown physiologic function. Previous studies demonstrated that the anti-Fas antibody mediated apoptosis in those cells sensitive to tumor necrosis factor (TNF) and, further, triggered the co-downregulation of tumor necrosis factor receptors (TNF-Rs). These findings led to speculation that Fas may be associated with TNF-Rs. The present studies were undertaken as an extension of our previous work on the obligate requirement for TNF in development and maintenance of cytotoxic lymphocytes and were designed to analyze the expression and consequences of Fas engagement in these cells. Herein, we demonstrate that, in contrast to TNF-R expression, both resting and IL-2-activated lymphocytes express Fas. In accordance with previous studies using tumor cell lines, lymphocytes rapidly downregulate TNF-Rs after treatment with anti-Fas. The ability of anti-Fas to mediate apoptotic cell death in lymphocytes, however, was dependent upon the status of cellular activation. For example, lymphocytes activated in IL-2 for longer than 4 days underwent rapid DNA fragmentation and cell death after anti-Fas treatment. Despite their expression of Fas, nonactivated lymphocytes and those activated for periods less than 4 days were refractory to antibody-mediated cell killing. Because anti-Fas-mediated lethality is selective for chronically activated lymphocytes, Fas may prove to be an appropriate target for immunosuppressive intervention.     

Adenoviral-mediated transfer of the TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces tumor cell apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells. The results presented in this study demonstrate that introduction of the human TRAIL gene into TRAIL-sensitive tumor cells using an adenoviral vector leads to the rapid production and expression of TRAIL protein, and subsequent death of the tumor cells. Tumor cell death was mediated by an apoptotic mechanism, as evidenced by the activation of caspase-8, cleavage of poly(ADP-ribose) polymerase, binding of annexin V, and inhibition by caspase inhibitor zVAD-fmk. These results define a novel method of using TRAIL as an antitumor therapeutic, and suggest the potential use for an adenovirus-encoding TRAIL as a method of gene therapy for numerous cancer types in vivo.     

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.     

Death of HT29 adenocarcinoma cells induced by TNF family receptor activation is caspase-independent and displays features of both apoptosis and necrosis

The HT29 adenocarcinoma is a common model of epithelial cell differentiation and colorectal cancer and its death is an oft-analyzed response to TNF family receptor signaling. The death event itself remains poorly characterized and here we have examined the involvement of caspases using pan-caspase inhibitors. zVAD-fmk did not block death of HT29 cells in response to activation of the Fas, TRAIL, TNF, TWEAK and LTbeta receptors. The secondary induction of TNF or the other known bona fide death inducing ligands did not account for death following LTbeta receptor activation indicating that TNF family receptors can trigger a caspase-independent death pathway regardless of the presence of canonical death domains in the receptor. To provide a frame of reference, the phenotype of HT29 death was compared to four other TNF family receptor triggered death events; Fas induced Jurkat cell apoptosis, TNF/zVAD induced L929 fibroblast necrosis, TNF induced death of WEHI 164 fibroblastoid cells and TNF/zVAD induced U937 death. The death of HT29 and U937 cells under these conditions is an intermediate form with both necrotic and apoptotic features. The efficient coupling of TNF receptors to a caspase-independent death event in an epithelial cell suggests an alternative approach to cancer therapy.     

Measles virus targets DC-SIGN to enhance dendritic cell infection

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in cis. Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

Cytolysis of tumor necrosis factor (TNF)-resistant tumor targets. Differential cytotoxicity of monocytes activated by the interferons, IL-2, and TNF

Herein we demonstrate that IFN-alpha, IFN-gamma, and IL-2 can induce human peripheral blood monocyte-mediated lysis of tumor cells that are resistant to both the direct effects of TNF and to monocytes activated by TNF. Monocytes activated by TNF kill only TNF-sensitive tumor targets, whereas those activated by IFN and IL-2 can lyse both TNF-sensitive and TNF-resistant tumor targets. Monocyte cytotoxicity against TNF-sensitive lines induced by the IFN, IL-2, or TNF can be completely abrogated by the addition of anti-TNF antibodies. In contrast, anti-TNF antibodies have no effect on IFN- or IL-2-induced monocyte cytotoxicity against TNF resistant targets, confirming non-TNF-mediated lysis induced by lymphokine-activated monocytes. Neither induction of TNF receptors by IFN-gamma nor inhibition of RNA synthesis by actinomycin D increased the susceptibility of TNF-resistant tumor targets to TNF-mediated monocyte cytotoxicity. Thus, non-TNF-mediated modes of monocyte cytotoxicity are induced by IFN and IL-2, but not by TNF, indicating that different cytotoxic mechanisms are responsible for the lysis of TNF-sensitive and TNF-resistant tumor cells. In addition, these findings also suggest that TNF-sensitive lines are susceptible only to TNF-mediated killing and apparently insensitive to non-TNF-mediated monocyte cytotoxicity.     

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.     

The C-type lectin macrophage galactose-type lectin impedes migration of immature APCs

Dendritic cells (DCs) are the most potent APCs of the immune system that seed the peripheral tissues and lymphoid organs. In an immature state, DCs sample their surroundings for incoming pathogens. Upon Ag encounter, DCs mature and migrate to the lymph node to induce adaptive immune responses. The C-type macrophage galactose-type lectin (MGL), expressed in immature DCs, mediates binding to glycoproteins carrying GalNAc moieties. In the present study, we demonstrate that MGL ligands are present on the sinusoidal and lymphatic endothelium of lymph node and thymus, respectively. MGL binding strongly correlated with the expression of the preferred MGL ligand, alpha-GalNAc-containing glycan structures, as visualized by staining with the alpha-GalNAc-specific snail lectin Helix pomatia agglutinin. MGL(+) cells were localized in close proximity of the endothelial structures that express the MGL ligand. Strikingly, instead of inducing migration, MGL mediated retention of human immature DCs, as blockade of MGL interactions enhanced DC trafficking and migration. Thus, MGL(+) DCs are hampered in their migratory responses and only upon maturation, when MGL expression is abolished; these DCs will be released from their MGL-mediated restraints.