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Siglec receptors and their ligand, sialic acids, are a novel immune checkpoint for immunotherapy.Cancer cells increase their expression of sialic acid-containing glycans.Cancer-associated fibroblasts have also been shown to express sialic acids and are a new area of research.The sialic acid/Siglec checkpoint drives myeloid cells to a protumorigenic phenotype.Clinical trials targeting the sialic acid–Siglec axis are underway.

Cellulose nanocrystal and gold nanoparticles are assembled, in a unique way, to yield a novel modular glyconanomaterial whose surface is then easily engineered with one or two different headgroups, by exploiting a robust click chemistry route. We demonstrate the potential of this approach by conjugating monosaccharide headgroups to the glyconanomaterial and show that the sugars retain their binding capability to C-type lectin receptors, as also directly visualized by cryo-TEM.

The gut microbiota guide the development of the host immune system by setting a systemic threshold for immune activation. Lipopolysaccharides (LPSs) from gut bacteria are able to trigger systemic and local proinflammatory and immunomodulatory responses, and this capability strongly relies on their fine structures. Up to now, only a few LPS structures from gut commensals have been elucidated; therefore, the molecular motifs that may be important for LPS–mammalian cell interactions at the gut level are still obscure. Here, we report on the full structure of the LPS isolated from one of the prominent species of the genus Bacteroides, Bacteroides vulgatus. The LPS turned out to consist of a particular chemical structure based on hypoacylated and mono-phosphorylated lipid A and with a galactofuranose-containing core oligosaccharide and an O-antigen built up of mannose and rhamnose. The evaluation of the immunological properties of this LPS on human in vitromodels revealed a very interesting capability to produce anti-inflammatory cytokines and to induce a synergistic action of MD-2/TLR4- and TLR2-mediated signaling pathways.

Dendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation of DCs is key, as well as the production of cytokines that help to accomplish T cell responses. DC-SIGN is a frequently exploited PRR that can effectively be targeted with mannosylated antigens to enhance the induction of antigen-specific T cells. The natural O-mannosidic linkage is susceptible to enzymatic degradation, and its chemical sensitivity complicates the synthesis of mannosylated antigens. For this reason, (oligo)mannosides are generally introduced in a late stage of the antigen synthesis, requiring orthogonal conjugation handles for their attachment. To increase the stability of the mannosides and streamline the synthesis of mannosylated peptide antigens, we here describe the development of an acid-stable C-mannosyl lysine, which allows for the inline introduction of mannosides during solid-phase peptide synthesis (SPPS). The developed amino acid has been successfully used for the assembly of both small ligands and peptide antigen conjugates comprising an epitope of the gp100 melanoma-associated antigen and a TLR7 agonist for DC activation. The ligands showed similar internalization capacities and binding affinities as the O-mannosyl analogs. Moreover, the antigen conjugates were capable of inducing maturation, stimulating the secretion of pro-inflammatory cytokines, and providing enhanced gp100 presentation to CD8+and CD4+T cells, similar to their O-mannosyl counterparts. Our results demonstrate that the C-mannose lysine is a valuable building block for the generation of anticancer peptide-conjugate vaccine modalities.

Tumour growth is accompanied by tumour evasion of the immune system, a process that is facilitated by immune checkpoint molecules such as programmed cell death protein 1 (PD1). However, the role of tumour glycosylation in immune evasion has mostly been overlooked, despite the fact that aberrant tumour glycosylation alters how the immune system perceives the tumour and can also induce immunosuppressive signalling through glycan-binding receptors. As such, specific glycan signatures found on tumour cells can be considered as a novel type of immune checkpoint. In parallel, glycosylation of tumour proteins generates neo-antigens that can serve as targets for tumour-specific T cells. In this Opinion article, we highlight how the tumour 'glyco-code' modifies immunity and suggest that targeting glycans could offer new therapeutic opportunities.

Splenic CD169+macrophages are located in the marginal zone to efficiently capture blood-borne pathogens. Here, we investigate the requirements for the induction of CD8+T cell responses by antigens (Ags) bound by CD169+macrophages. Upon Ag targeting to CD169+macrophages, we show that BATF3-dependent CD8α+dendritic cells (DCs) are crucial for DNGR-1-mediated cross-priming of CD8+T cell responses. In addition, we demonstrate that CD169, a sialic acid binding lectin involved in cell-cell contact, preferentially binds to CD8α+DCs and that Ag transfer to CD8α+DCs and subsequent T cell activation is dependent on the sialic acid-binding capacity of CD169. Finally, functional CD169 mediates optimal CD8+T cell responses to modified vaccinia Ankara virus infection. Together, these data indicate that the collaboration of CD169+macrophages and CD8α+DCs for the initiation of effective CD8+T cell responses is facilitated by binding of CD169 to sialic acid containing ligands on CD8α+DCs.

A vaccine platform developed from a synthetic polymeric glyco-adjuvant and reversibly conjugated to an antigen was shown to target dendritic cells leading to cellular and humoral immune response against malaria.

Salivary agglutinin (SAG), also known as gp340 or SALSA, is a glycoprotein encoded by the Deleted in Malignant Brain Tumours 1gene and is abundantly present in human saliva. SAG aggregates bacteria and viruses, thereby promoting their clearance from the oral cavity. The mucosa lining the oral cavity contains dendritic cells (DC) and Langerhans cells (LC), which express the C-type lectin receptors (CLR) DC-SIGN and Langerin, respectively. Both DC-SIGN and Langerin recognise mannose and fucose carbohydrate structures on pathogens and self-glycoproteins to regulate immunity and homeostasis. The purpose of this study was to investigate whether SAG interacts with these CLR and whether this interferes with the binding to oral pathogens. We show that whole parotid saliva and SAG, when coated to microplates, strongly interact with DC-SIGN and Langerin, probably via mannose and fucose structures. Also, primary human DC and LC bind parotid saliva and SAG via DC-SIGN and Langerin, respectively. Furthermore, SAG binding to DC-SIGN or Langerin prevented binding to the micro-organisms Candida albicansand Escherichia coliwhich express mannose and fucose-containing glycan structures. Thus, binding of saliva glycoprotein SAG to DC-SIGN and Langerin may inhibit pathogen-DC/LC interactions, and could prove to be a new immunomodulatory mechanism of SAG.

AbstractDietary intervention using nondigestible oligosaccharides, short-chain galacto-oligosaccharides (scGOS)/long-chain fructo-oligosaccharides (lcFOS), in combination with Bifidobacterium breveM-16V prevents allergic disease involving galectin-9. In addition, apical TLR9 signaling contributes to intestinal homeostasis. We studied the contribution of galectin-9 secreted by intestinal epithelial cells (IEC; HT-29 and T84) in Th1 and regulatory T-cell (Treg) polarization in vitro. IEC were grown in transwell filters, cocultured with CD3/CD28-activated human peripheral blood mononuclear cells (PBMC) and apically exposed to genomic DNA derived from B. breveM-16V or synthetic TLR9 ligand in the absence or presence of scGOS/lcFOS. Cytokine production and T-cell phenotype were determined and galectin expression by IEC was assessed. Galectin-9 was neutralized using lactose or a TIM-3-Fc fusion protein. IEC exposed to DNA from B. breveM-16V or TLR9 ligand in the presence of scGOS/lcFOS enhanced IFN-γ secretion by PBMC and increased the percentage of Th1 and Tregcells. Expression and secretion of galectin-9 by IEC was increased and neutralization of galectin-9 prevented the induction of IFN-γ secretion and also suppressed the production of IL-10 by PBMC. Furthermore, we show that galectin-9 induces Tregand Th1 polarization through interaction with antigen-presenting cells. Our findings show that galectin-9 secreted by IEC apically exposed to TLR9 ligand in the presence of scGOS/lcFOS is involved in Th1 and Tregpolarization and may be a promising target to prevent or treat allergic disease.Copyright © 2013 S. Karger AG, Basel

The generation of effector CD8+T cells with lytic capacity is crucial for tumor control. Dendritic cells (DCs) provide important signals to promote naive CD8+T cell priming and activation of effector T cells. Here, we report that the Notch pathway has an important role in both these processes in human CD8+T cells. Activated monocyte-derived DCs express Notch ligands Jagged1 and Delta-like4, whereas naive CD8+T cells express Notch2. The role for Notch signaling in CD8+T cell priming was determined using an ex-vivo model system in which tumor antigen–specific primary CD8+T cell responses were measured. Inhibition of Notch using γ-secretase inhibitors or soluble Delta-like4-Fc during activation reduced expansion of antigen-specific CD8+T cells, which was mirrored by decreased frequencies of interferon (IFN)γ-, tumor necrosis factor-α–, and granzymeB-producing CD8+T cells. Moreover, T cells primed when Notch signaling was prevented are functionally low-avidity T cells. In addition, Notch partially regulates established effector T cell function. Activation-induced Notch signaling is needed for IFNγ release but not for cytolytic activity. These data indicate that Notch signaling controls human CD8+T cell priming and also influences effector T cell functions. This may provide important information for designing new immunotherapies for treatment of cancer.

The generation of effector CD8+ T cells with lytic capacity is crucial for tumor control. Dendritic cells (DCs) provide important signals to promote naive CD8+ T cell priming and activation of effector T cells. Here, we report that the Notch pathway has an important role in both these processes in human CD8+ T cells. Activated monocyte-derived DCs express Notch ligands Jagged1 and Delta-like4, whereas naive CD8+ T cells express Notch2. The role for Notch signaling in CD8+ T cell priming was determined using an ex-vivo model system in which tumor antigen–specific primary CD8+ T cell responses were measured. Inhibition of Notch using γ-secretase inhibitors or soluble Delta-like4-Fc during activation reduced expansion of antigen-specific CD8+ T cells, which was mirrored by decreased frequencies of interferon (IFN)γ-, tumor necrosis factor-α–, and granzymeB-producing CD8+ T cells. Moreover, T cells primed when Notch signaling was prevented are functionally low-avidity T cells. In addition, Notch partially regulates established effector T cell function. Activation-induced Notch signaling is needed for IFNγ release but not for cytolytic activity. These data indicate that Notch signaling controls human CD8+ T cell priming and also influences effector T cell functions. This may provide important information for designing new immunotherapies for treatment of cancer.

ABSTRACTCarbohydrate mimicry between Campylobacter jejunilipooligosaccharides (LOS) and host neural gangliosides plays a crucial role in the pathogenesis of Guillain-Barré syndrome (GBS). Campylobacter jejuniLOS may mimic various gangliosides, which affects the immunogenicity and the type of neurological deficits in GBS patients. Previous studies have shown the interaction of LOS with sialic acid-specific siglec receptors, although the functional consequences remain unknown. Cells that express high levels of siglecs include dendritic cells (DCs), which are crucial for initiation and differentiation of immune responses. We confirm that α2,3-sialylated GD1a/GM1a mimic and α2,8-sialylated GD1c mimic LOS structures interact with recombinant Sn and siglec-7, respectively. Although the linkage of the terminal sialic acid of LOS did not regulate expression of DC maturation markers, it displayed clear opposite expression levels of interleukin-12 (IL-12) and OX40L, molecules involved in DC-mediated Th cell differentiation. Accordingly, targeting DC-expressed siglec-7 with α2,8-linked sialylated LOS resulted in Th1 responses, whereas Th2 responses were induced by targeting with LOS containing α2,3-linked sialic acid. Thus, our data demonstrate for the first time that depending on the sialylated composition of Campylobacter jejuniLOS, specific Th differentiation programs are initiated, possibly through targeting of distinct DC-expressed siglecs.

Carbohydrate mimicry between Campylobacter jejuni lipooligosaccharides (LOS) and host neural gangliosides plays a crucial role in the pathogenesis of Guillain-Barré syndrome (GBS). Campylobacter jejuni LOS may mimic various gangliosides, which affects the immunogenicity and the type of neurological deficits in GBS patients. Previous studies have shown the interaction of LOS with sialic acid-specific siglec receptors, although the functional consequences remain unknown. Cells that express high levels of siglecs include dendritic cells (DCs), which are crucial for initiation and differentiation of immune responses. We confirm that α2,3-sialylated GD1a/GM1a mimic and α2,8-sialylated GD1c mimic LOS structures interact with recombinant Sn and siglec-7, respectively. Although the linkage of the terminal sialic acid of LOS did not regulate expression of DC maturation markers, it displayed clear opposite expression levels of interleukin-12 (IL-12) and OX40L, molecules involved in DC-mediated Th cell differentiation. Accordingly, targeting DC-expressed siglec-7 with α2,8-linked sialylated LOS resulted in Th1 responses, whereas Th2 responses were induced by targeting with LOS containing α2,3-linked sialic acid. Thus, our data demonstrate for the first time that depending on the sialylated composition of Campylobacter jejuni LOS, specific Th differentiation programs are initiated, possibly through targeting of distinct DC-expressed siglecs.

Dendritic cells (DCs) are essential for the early events of human immunodeficiency virus (HIV) infection. Model systems of HIV sexual transmission have shown that DCs expressing the DC-specific C-type lectin DC-SIGN capture and internalize HIV at mucosal surfaces and efficiently transfer HIV to CD4+ T cells in lymph nodes, where viral replication occurs. Upon DC–T cell clustering, internalized HIV accumulates on the DC side at the contact zone (infectious synapse), between DCs and T cells, whereas HIV receptors and coreceptors are enriched on the T cell side. Viral concentration at the infectious synapse may explain, at least in part, why DC transmission of HIV to T cells is so efficient. Here, we have investigated the role of DC-SIGN on primary DCs in X4 HIV-1 capture and transmission using small interfering RNA–expressing lentiviral vectors to specifically knockdown DC-SIGN. We demonstrate that DC-SIGN− DCs internalize X4 HIV-1 as well as DC-SIGN+ DCs, although binding of virions is reduced. Strikingly, DC-SIGN knockdown in DCs selectively impairs infectious synapse formation between DCs and resting CD4+ T cells, but does not prevent the formation of DC–T cells conjugates. Our results demonstrate that DC-SIGN is required downstream from viral capture for the formation of the infectious synapse between DCs and T cells. These findings provide a novel explanation for the role of DC-SIGN in the transfer and enhancement of HIV infection from DCs to T cells, a crucial step for HIV transmission and pathogenesis.

The human gastric pathogen Helicobacter pylori spontaneously switches lipopolysaccharide (LPS) Lewis (Le) antigens on and off (phase-variable expression), but the biological significance of this is unclear. Here, we report that Le+ H. pylori variants are able to bind to the C-type lectin DC-SIGN and present on gastric dendritic cells (DCs), and demonstrate that this interaction blocks T helper cell (Th)1 development. In contrast, Le− variants escape binding to DCs and induce a strong Th1 cell response. In addition, in gastric biopsies challenged ex vivo with Le+ variants that bind DC-SIGN, interleukin 6 production is decreased, indicative of increased immune suppression. Our data indicate a role for LPS phase variation and Le antigen expression by H. pylori in suppressing immune responses through DC-SIGN.

In the paracortex of lymph nodes, cellular immune responses are generated against antigens captured in peripheral tissues by dendritic cells (DCs). DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), a C-type lectin exclusively expressed by DCs, functions as an antigen receptor as well as an adhesion receptor. A functional homologue of DC-SIGN, L-SIGN (liver/lymph node-SIGN, also called DC-SIGN-related), is expressed by liver sinus endothelial cells. In lymph nodes, both DC-SIGN and L-SIGN are expressed. In this study, we analyzed the distribution of these two SIGN molecules in detail in both normal and immunoreactive lymph nodes. DC-SIGN is expressed by mature DCs in paracortical areas and in addition by DCs with an immature phenotype in the outer zones of the paracortex. L-SIGN expression was also detected in the outer zones on sinus endothelial cells characterized by their expression of the lymphatic endothelial markers LYVE-1 and CLEVER-1. During both cellular and humoral immune responses changes in the amount of DC-SIGN+immature and mature DCs and L-SIGN+endothelial cells were observed, indicating that the influx or proliferation of these cells is dynamically regulated.

Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83+DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR+/DC-SIGN+APCs further phenotyped as CD14+/CD4+/CD68+/−/CD83−/CD25−. These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209+cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN+decidual APCs were observed in situto have intimate contact with CD56+/CD16−/ICAM-3+decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14+/DC-SIGN+decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14+/DC-SIGN+decidual cells into CD25+/CD83+mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset.

Mycobacterium tuberculosis represents a world-wide health risk and immunosuppression is a particular problem in M. tuberculosis infections. Although macrophages are primarily infected, dendritic cells (DCs) are important in inducing cellular immune responses against M. tuberculosis. We hypothesized that DCs represent a target for M. tuberculosis and that the observed immuno-suppression results from modulation of DC functions. We demonstrate that the DC-specific C-type lectin DC-SIGN is an important receptor on DCs that captures and internalizes intact Mycobacterium bovis bacillus Calmette-Guérin (BCG) through the mycobacterial cell wall component ManLAM. Antibodies against DC-SIGN block M. bovis BCG infection of DCs. ManLAM is also secreted by M. tuberculosis–infected macrophages and has been implicated as a virulence factor. Strikingly, ManLAM binding to DC-SIGN prevents mycobacteria- or LPS-induced DC maturation. Both mycobacteria and LPS induce DC maturation through Toll-like receptor (TLR) signaling, suggesting that DC-SIGN, upon binding of ManLAM, interferes with TLR-mediated signals. Blocking antibodies against DC-SIGN reverse the ManLAM-mediated immunosuppressive effects. Our results suggest that M. tuberculosis targets DC-SIGN both to infect DCs and to down-regulate DC-mediated immune responses. Moreover, we demonstrate that DC-SIGN has a broader pathogen recognition profile than previously shown, suggesting that DC-SIGN may represent a molecular target for clinical intervention in infections other than HIV-1.

Dentritic cells (DC) as antigen-presenting cells are most likely responsible for regulation of abnormal T cell activation in Crohn's disease (CD), a chronic inflammatory bowel disease. Wehave analyzed the expression of activation and maturation markers on DC in the colon mucosa from patients with CD compared with normal colon, using immunohistochemical techniques. We found two distinct populations of DC present in CD patients: a DC-specific ICAM-3 grabbing non-integrin (DC-SIGN)+ population that was present scattered throughout the mucosa, and a CD83+ population that was present in aggregated lymphoid nodules and as single cells in the lamina propria. In normal colon the number of DC-SIGN+ DC was lower and CD83+ DC were detected only in very few solitary lymphoid nodules. Co-expression of activation markers and cytokine synthesis was analyzed with three-color confocal laser scanning microscopy analysis. CD80 expression was enhanced on the majority of DC-SIGN+ DC in CD patients, whereas only a proportion of the CD83+ DC co-expressed CD80 in CD as well as in normal tissue. Surprisingly, IL-12 and IL-18were only detected in DC-SIGN+ DC and not in CD83+ DC. A similar pattern of cytokine production was observed in normal colon albeit to a much lesser extent. The characteristics ofthese in-situ-differentiated DC markedly differ from the in-vitro-generated DC that simultaneously express DC-SIGN, CD83 and cytokines.

Antigen-presenting cells are localized in essentially every tissue, where they operate at the interface of innate and acquired immunity by capturing pathogens and presenting pathogen-derived peptides to T cells. C-type lectins are important pathogen recognition receptors and the C-type lectin, dendritic cell–specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), is unique in that, in addition to pathogen capture, it regulates adhesion processes such as DC trafficking and T-cell synapse formation. We have isolated a murine homologue of DC-SIGN that is identical to the previously reported murine homologue mSIGNR1. mSIGNR1 is more closely related to the human DC-SIGN homologue L-SIGN than to DC-SIGN itself because mSIGNR1 is specifically expressed by liver sinusoidal endothelial cells, similar to L-SIGN, and not by DCs. Moreover, mSIGNR1 is also expressed by medullary and subcapsular macrophages in lymph nodes and by marginal zone macrophages (MZMs) in the spleen. Strikingly, these MZMs are in direct contact with the bloodstream and efficiently capture specific polysaccharide antigens present on the surface of encapsulated bacteria. We have investigated the in vivo function of mSIGNR1 on MZMs in spleen. We demonstrate here that mSIGNR1 functions in vivo as a pathogen recognition receptor on MZMs that capture blood-borne antigens, which are rapidly internalized and targeted to lysosomes for processing. Moreover, the antigen capture is completely blocked in vivo by the blocking mSIGNR1-specific antibodies. Thus, mSIGNR1, a murine homologue of DC-SIGN, is important in the defense against pathogens and this study will facilitate further investigations into the in vivo function of DC-SIGN and its homologues.

Antigen-presenting cells are localized in essentially every tissue, where they operate at the interface of innate and acquired immunity by capturing pathogens and presenting pathogen-derived peptides to T cells. C-type lectins are important pathogen recognition receptors and the C-type lectin, dendritic cell–specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), is unique in that, in addition to pathogen capture, it regulates adhesion processes such as DC trafficking and T-cell synapse formation. We have isolated a murine homologue of DC-SIGN that is identical to the previously reported murine homologue mSIGNR1. mSIGNR1 is more closely related to the human DC-SIGN homologue L-SIGN than to DC-SIGN itself because mSIGNR1 is specifically expressed by liver sinusoidal endothelial cells, similar to L-SIGN, and not by DCs. Moreover, mSIGNR1 is also expressed by medullary and subcapsular macrophages in lymph nodes and by marginal zone macrophages (MZMs) in the spleen. Strikingly, these MZMs are in direct contact with the bloodstream and efficiently capture specific polysaccharide antigens present on the surface of encapsulated bacteria. We have investigated the in vivo function of mSIGNR1 on MZMs in spleen. We demonstrate here that mSIGNR1 functions in vivo as a pathogen recognition receptor on MZMs that capture blood-borne antigens, which are rapidly internalized and targeted to lysosomes for processing. Moreover, the antigen capture is completely blocked in vivo by the blocking mSIGNR1-specific antibodies. Thus, mSIGNR1, a murine homologue of DC-SIGN, is important in the defense against pathogens and this study will facilitate further investigations into the in vivo function of DC-SIGN and its homologues.

VLA-4 and LFA-1 are the major vascular integrins expressed on circulating lymphocytes. Previous studies suggested that intact cholesterol rafts are required for integrin adhesiveness in different leukocytes. We found the alpha(4) integrins VLA-4 and alpha(4)beta(7) as well as the LFA-1 integrin to be excluded from rafts of human peripheral blood lymphocytes. Disruption of cholesterol rafts with the chelator methyl-beta-cyclodextrin did not affect the ability of these lymphocyte integrins to generate high avidity to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow. In contrast, cholesterol extraction abrogated rapid chemokine triggering of alpha(4)-integrin-dependent peripheral blood lymphocytes adhesion, a process tightly regulated by G(i)-protein activation of G protein-coupled chemokine receptors (GPCR). Strikingly, stimulation of LFA-1 avidity to intercellular adhesion molecule 1 (ICAM-1) by the same chemokines, although G(i)-dependent, was insensitive to raft disruption. Our results suggest that alpha(4) but not LFA-1 integrin avidity stimulation by chemokines involves rapid chemokine-induced GPCR rearrangement that takes place at cholesterol raft platforms upstream to G(i) signaling. Our results provide the first evidence that a particular chemokine/GPCR pair can activate different integrins on the same cell using distinct G(i) protein-associated machineries segregated within defined membrane compartments.

The dendritic cell (DC)–specific molecule DC-SIGN is a receptor for the HIV-1 envelope glycoprotein gp120 and is essential for the dissemination of HIV-1. DC-SIGN is expressed by DCs, both monocyte-derived DCs and DCs in several tissues, including mucosa and lymph nodes. To identify a DC-SIGN+ DC in blood that may be involved in HIV-1 infection through blood, we have analyzed the expression of DC-SIGN in human blood cells. Here we describe the characterization of a subset of DCs in human blood, isolated from T-/NK-/B-cell–depleted peripheral blood mononuclear cells (PBMCs) on the basis of expression of DC-SIGN. This subset coexpresses CD14, CD16, and CD33 and is thus of myeloid origin. In contrast to CD14+ monocytes, DC-SIGN+ blood cells display a DC-like morphology and express markers of antigen-presenting cells, including CD1c, CD11b, CD11c, CD86, and high levels of major histocompatibility complex (MHC) class I and II molecules. This DC population differs from other described CD14−blood DC subsets. Functionally, DC-SIGN+ blood DCs are able to stimulate proliferation of allogeneic T cells and can produce tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) upon activation with lipopolysaccharide (LPS). When they encounter HIV-1, low amounts of these blood DC-SIGN+ DCs enhance infection of T lymphocytes in trans, whereas blood monocytes and CD14−blood DCs are not capable of transmitting HIV-1. Therefore DC-SIGN+ blood DCs can be the first target for HIV-1 upon transmission via blood; they can capture minute amounts of HIV-1 through DC-SIGN and transfer HIV-1 to infect target T cells in trans.

The dendritic cell (DC)–specific molecule DC-SIGN is a receptor for the HIV-1 envelope glycoprotein gp120 and is essential for the dissemination of HIV-1. DC-SIGN is expressed by DCs, both monocyte-derived DCs and DCs in several tissues, including mucosa and lymph nodes. To identify a DC-SIGN+DC in blood that may be involved in HIV-1 infection through blood, we have analyzed the expression of DC-SIGN in human blood cells. Here we describe the characterization of a subset of DCs in human blood, isolated from T-/NK-/B-cell–depleted peripheral blood mononuclear cells (PBMCs) on the basis of expression of DC-SIGN. This subset coexpresses CD14, CD16, and CD33 and is thus of myeloid origin. In contrast to CD14+monocytes, DC-SIGN+blood cells display a DC-like morphology and express markers of antigen-presenting cells, including CD1c, CD11b, CD11c, CD86, and high levels of major histocompatibility complex (MHC) class I and II molecules. This DC population differs from other described CD14−blood DC subsets. Functionally, DC-SIGN+blood DCs are able to stimulate proliferation of allogeneic T cells and can produce tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) upon activation with lipopolysaccharide (LPS). When they encounter HIV-1, low amounts of these blood DC-SIGN+DCs enhance infection of T lymphocytes in trans, whereas blood monocytes and CD14−blood DCs are not capable of transmitting HIV-1. Therefore DC-SIGN+blood DCs can be the first target for HIV-1 upon transmission via blood; they can capture minute amounts of HIV-1 through DC-SIGN and transfer HIV-1 to infect target T cells in trans.

The β2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34+ cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti–LFA-1 antibodies completely prevent the IL-8–induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-β2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)–induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the α chain of LFA-1 or against the α chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area–forming cells in vitro was significantly higher after mobilization with anti–LFA-1 antibodies followed by 5 μg G-CSF for 5 days than with G-CSF alone (119 ± 34 days vs 17 ± 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF–induced mobilization, suggesting that the enhancing effect required an interaction of the β2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1–deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1–mediated adhesion.

The β2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34+cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti–LFA-1 antibodies completely prevent the IL-8–induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-β2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)–induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the α chain of LFA-1 or against the α chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area–forming cells in vitro was significantly higher after mobilization with anti–LFA-1 antibodies followed by 5 μg G-CSF for 5 days than with G-CSF alone (119 ± 34 days vs 17 ± 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF–induced mobilization, suggesting that the enhancing effect required an interaction of the β2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1–deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1–mediated adhesion.

Dendritic cells (DC) are present in essentially every tissue where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen‐derived peptides to T cells. It is becoming clear that not all C‐type lectins on DC serve as antigen receptors recognizing pathogens through carbohydrate structures. The C‐type lectin DC‐SIGN is unique in that it regulates adhesion processes, such as DC trafficking and T‐cell synapse formation, as well as antigen capture. Moreover, even though several C‐type lectins have been shown to bind HIV‐1, DC‐SIGN does not only capture HIV‐1 but also protects it in early endosomes allowing HIV‐1 transport by DC to lymphoid tissues, where it enhances transinfection of T cells. Here we discuss the carbohydrate/protein recognition profile and other features of DC‐SIGN that contribute to the potency of DC to control immunity.

The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV-1 to T cells. DC-SIGN is also important in the initiation of immune responses by regulating DC-T cell interactions through intercellular adhesion molecule 3 (ICAM-3). We have characterized the mechanism of ligand binding by DC-SIGN and identified the crucial amino acids involved in this process. Strikingly, the HIV-1 gp120 binding site in DC-SIGN is different from that of ICAM-3, consistent with the observation that glycosylation of gp120, in contrast to ICAM-3, is not crucial to the interaction with DC-SIGN. A specific mutation in DC-SIGN abrogated ICAM-3 binding, whereas the HIV-1 gp120 interaction was unaffected. This DC-SIGN mutant captured HIV-1 and infected T cells in transas efficiently as wild-type DC-SIGN, demonstrating that ICAM-3 binding is not necessary for HIV-1 transmission. This study provides a basis for the design of drugs that inhibit or alter interactions of DC-SIGN with gp120 but not with ICAM-3 or vice versa and that have a therapeutic value in immunological diseases and/or HIV-1 infections.

In the primary sequence of the integrin β subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin β2NTR and mid-region, we constructed β2/β7chimeras in which the NTR, I-like domain, and the mid-region of the β2subunit were replaced by those of β7. Changing either the β2NTR or mid-region, but not the I-like domain to that of β7did not affect LFA-1 (αLβ2) formation and surface expression. Thus, the specificity of αLβ2pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-β2mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the β2NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the β2NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

Various pathologic conditions, such as hemorrhage, hemolysis and cell injury, are characterized by the release of large amounts of heme. Recently, it was demonstrated that heme oxygenase (HO), the heme-degrading enzyme, and heme are able to modulate adhesion molecule expression in vitro. In the present study, the effects of heme and HO on inflammation in mice were analyzed by monitoring the biodistribution of radiolabeled liposomes and leukocytes in conjunction with immunohistochemistry. Small liposomes accumulate in inflamed tissues by diffusion because of locally enhanced vascular permeability, whereas leukocytes actively migrate into inflammatory areas through specific adhesive interactions with the endothelium and chemotaxis. Exposure to heme resulted in a dramatic increase in liposome accumulation in the pancreas, but also intestines, liver, and spleen exhibited significantly increased vascular permeability. Similarly, intravenously administered heme caused an enhanced influx of radiolabeled leukocytes into these organs. Immunohistochemical analysis showed differential up-regulation of the adhesion molecules ICAM-1, P-selectin, and fibronectin in liver and pancreas in heme-treated animals. Heme-induced adhesive properties were accompanied by a massive influx of granulocytes into these inflamed tissues, suggesting an important contribution to the pathogenesis of inflammatory processes. Moreover, inhibition of HO activity exacerbated heme-induced granulocyte infiltration. Here it is demonstrated for the first time that heme induces increased vascular permeability, adhesion molecule expression, and leukocyte recruitment in vivo, whereas HO antagonizes heme-induced inflammation possibly through the down-modulation of adhesion molecules.

Various pathologic conditions, such as hemorrhage, hemolysis and cell injury, are characterized by the release of large amounts of heme. Recently, it was demonstrated that heme oxygenase (HO), the heme-degrading enzyme, and heme are able to modulate adhesion molecule expression in vitro. In the present study, the effects of heme and HO on inflammation in mice were analyzed by monitoring the biodistribution of radiolabeled liposomes and leukocytes in conjunction with immunohistochemistry. Small liposomes accumulate in inflamed tissues by diffusion because of locally enhanced vascular permeability, whereas leukocytes actively migrate into inflammatory areas through specific adhesive interactions with the endothelium and chemotaxis. Exposure to heme resulted in a dramatic increase in liposome accumulation in the pancreas, but also intestines, liver, and spleen exhibited significantly increased vascular permeability. Similarly, intravenously administered heme caused an enhanced influx of radiolabeled leukocytes into these organs. Immunohistochemical analysis showed differential up-regulation of the adhesion molecules ICAM-1, P-selectin, and fibronectin in liver and pancreas in heme-treated animals. Heme-induced adhesive properties were accompanied by a massive influx of granulocytes into these inflamed tissues, suggesting an important contribution to the pathogenesis of inflammatory processes. Moreover, inhibition of HO activity exacerbated heme-induced granulocyte infiltration. Here it is demonstrated for the first time that heme induces increased vascular permeability, adhesion molecule expression, and leukocyte recruitment in vivo, whereas HO antagonizes heme-induced inflammation possibly through the down-modulation of adhesion molecules.

The leukocyte-specific β2integrin lymphocyte function-associated antigen-1 (LFA-1) (αL/β2) mediates activation-dependent adhesion to intercellular adhesion molecule (ICAM)-1. In leukocytes, LFA-1 requires activation by intracellular messengers to bind ICAM-1. We observed malfunctioning of LFA-1 activation in leukemic T cells and K562-transfected cells. This defective inside-out integrin activation is only restricted to β2integrins, since β1integrins expressed in K562 readily respond to activation signals, such as phorbol 12-myristate 13-acetate. To unravel these differences in inside-out signaling between β1and β2integrins, we searched for amino acids in the β2cytoplasmic domain that are critical in the activation of LFA-1. We provide evidence that substitution of a single amino acid (L732R) in the β2cytoplasmic DLRE motif, creating the DRRE motif, is sufficient to completely restore PMA responsiveness of LFA-1 expressed in K562. In addition, an intact TTT motif in the C-terminal domain is necessary for the acquired PMA responsiveness. We observed that restoration of the PMA response altered neither LFA-1 affinity nor the phosphorylation status of LFA-1. In contrast, strong differences were observed in the capacity of LFA-1 to form clusters, which indicates that inside-out activation of LFA-1 strongly depends on cytoskeletal induced receptor reorganization that was induced by activation of the Ca2+-dependent protease calpain.

The discovery of dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) as a DC-specific ICAM-3 binding receptor that enhances HIV-1 infection of T cells in trans has indicated a potentially important role for adhesion molecules in AIDS pathogenesis. A related molecule called DC-SIGNR exhibits 77% amino acid sequence identity with DC-SIGN. The DC-SIGN and DC-SIGNR genes map within a 30-kb region on chromosome 19p13.2-3. Their strong homology and close physical location indicate a recent duplication of the original gene. Messenger RNA and protein expression patterns demonstrate that the DC-SIGN–related molecule is highly expressed on liver sinusoidal cells and in the lymph node but not on DCs, in contrast to DC-SIGN. Therefore, we suggest that a more appropriate name for the DC-SIGN–related molecule is L-SIGN, liver/lymph node–specific ICAM-3–grabbing nonintegrin. We show that in the liver, L-SIGN is expressed by sinusoidal endothelial cells. Functional studies indicate that L-SIGN behaves similarly to DC-SIGN in that it has a high affinity for ICAM-3, captures HIV-1 through gp120 binding, and enhances HIV-1 infection of T cells in trans. We propose that L-SIGN may play an important role in the interaction between liver sinusoidal endothelium and trafficking lymphocytes, as well as function in the pathogenesis of HIV-1.

Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, α4β1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM–ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM–ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.

Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis is poorly understood. LFA-1 is considered as a marker of late stage stem cell maturation when expressed on CD34+bone marrow cells. We observed that CD34+bone marrow cells express LFA-1, that based on LFA-1 expression several subpopulations can be distinguished, and that the level of expression appeared highly variable among different donors. Unanticipated, in time course experiments we observed that CD34+LFA-1−cells expressed LFA-1 within 24 hours upon culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA-1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, thus equipping cells with adequate adhesion receptors to migrate throughout the bone marrow. Interestingly, the expression of the LFA-1 specific activation epitope L16 on these cells is low, even after culture. This demonstrated that LFA-1 is not activated, as was confirmed by low adhesion to ICAM-1. Activation of adhesion molecules is induced by several growth factors. Indeed, we show here that an osteoblastic cell line under normal conditions does hardly produce hematopoietic growth factors but these are rapidly induced after stimulation. Such rapid induction endows the bone marrow stroma with the property to modulate the adhesive strength and enabling migration through the different environments within the stroma. Prove for such compartments within the bone marrow is provided by histological data.

Recently, we have demonstrated that antibodies that block the function of the ß2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P < .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)-/Rhodamine (Rho)- sorted cells, were examined. More than 95% of the Rho- cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho- LFA-1neg or up to 600 Rho-LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477–3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

Aberrant proliferation, differentiation, and/or migration of progenitors observed in various hematological malignancies may be caused by defects in expression and/or function of integrins. In this study, we have developed a new fluorescent beads adhesion assay that facilitates flow cytometric investigation of lymphocyte function-associated antigen 1 (LFA-1)– and very late activation antigen-4 (VLA-4)–mediated functional adhesion in B-lineage acute lymphoblastic leukemia (ALL) of both the CD10- and CD10+ (leukemic) cell population within one blood or bone marrow sample. Surprisingly, of the 20 B-lineage ALL patients investigated, 17 contained a leukemic cell population with LFA-1– and/or VLA-4–mediated adhesion defects. Five patients contained CD10+ cells that did not exhibit any LFA-1–mediated adhesion due to the lack of LFA-1 surface expression. The CD10+ cells from 10 ALL patients expressed LFA-1 that could not be activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA), whereas the CD10- cells expressed a functional LFA-1. Seven patients contained CD10+ cells that expressed a PMA-unresponsive form of VLA-4. The PMA unresponsiveness of the integrins LFA-1 and VLA-4 expressed by the CD10+ cells may be due to mutations in the integrins itself, in protein kinases, or in other intracellular molecules involved in integrin adhesion. These data clearly demonstrate the importance of investigating integrin function in addition to integrin surface expression. The strikingly high frequency (85%) of adhesion defects in ALL could suggest a causal relationship between integrin-mediated adhesion and B-lineage ALL.

To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (αLβ2), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete β2cytoplasmic tail and/or the conserved KVGFFKR sequence in the αLcytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.

Previously, we have shown that interleukin (IL)-8 induces the rapid (15 to 30 minutes) mobilization of hematopoietic progenitor cells (HPC) in mice. Because integrins are essential for adhesion and transendothelial migration of HPC, we studied the involvement of the ß2-integrin leukocyte function-associated antigen-1 (LFA-1) in IL-8–induced mobilization. After a single injection of blocking anti–LFA-1 antibodies, no mobilization of colony-forming cells was observed. In addition, when mice were pretreated with anti–LFA-1 or saline and subsequently injected with 30 µg of IL-8, mobilization of HPC was completely blocked. We showed that this was not due to anti–LFA-1 antibodies affecting colony formation, as addition of anti–LFA-1 antibodies to colony cultures in semisolid medium had no inhibitory activity. Also, anti-intercellular adhesion molecule (ICAM)-1 antibodies, directed to the main ligand of LFA-1 significantly inhibited the IL-8–induced mobilization. Furthermore, IL-1–induced mobilization was significantly inhibited by anti–LFA-1 antibodies. Because LFA-1 is reported to be expressed on more differentiated HPC, it was considered that the IL-8–induced mobilization of more primitive HPC would not be blocked by anti–LFA-1 antibodies. Transplantation of blood-derived mononuclear cells (MNC) from IL-8–mobilized animals pretreated with anti–LFA-1 antibodies protected only 25% of lethally irradiated recipient mice, whereas the radioprotection rate of control mice transplanted with MNC derived from IL-8-mobilized animals was 86% (P < .01). Anti-LFA–1 antibodies did not interfere with stem cell homing, as transplantation of IL-8-mobilized blood MNC, incubated in vitro with these antibodies resulted in 100% radioprotection. We conclude that anti–LFA-1 antibodies completely prevent the rapid mobilization of colony-forming cells and of cells with radioprotective capacity induced by IL-8. These results indicate a major role for the ß2-integrin LFA-1 in the IL-8–induced mobilization of hematopoietic stem cells.

Adult T-cell leukemia (ATL) is characterized by massive infiltration of circulating ATL cells into a variety of tissues, a finding often associated with poor prognosis. Leukocyte migration from circulation into tissue depends on integrin-mediated adhesion to endothelium, and integrins are tightly regulated by several stimuli, such as inflammatory chemokines. However, the exact mechanisms that enhance adherence of leukemic cells to the endothelium and infiltration into tissues remain to be fully understood. We investigated the mechanisms of extravasation of leukemic cells using ATL cells and report the following novel features of endogenous chemokine-induced adhesion of ATL cells to the endothelium. ATL cells spontaneously adhered to endothelial cells without exogenous stimulation. Integrin leukocyte function-associated antigen-1 (LFA-1) on ATL cells was spontaneously activated. ATL cells produced high amounts of chemokines, macrophage inflammatory protein-1a (MIP-1a), and MIP-1ß. Adhesion of ATL cells to endothelial cells and the expression of activated form of LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or anti–MIP-1a and MIP-1ß antibodies or transfection with antisense of MIP-1a or MIP-1ß. Spontaneous polymerization of cytoskeletal F-actin was observed in ATL cells, which was also inhibited by pertussis toxin and wortmannin. We propose that ATL cells adhere to endothelial cells through an adhesion cascade similar to normal leukocytes and that the chemokines produced by ATL cells are involved in triggering integrin LFA-1 through cytoskeletal rearrangement induced by G-protein–dependent activation of phosphoinositide 3-kinases in an autocrine manner. These events result in a strong adhesion of ATL cells to the endothelium and spontaneous transendothelial migration.

Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis remains obscure. Since LFA-1 is predominantly expressed by leukocytes, it is considered as a marker of late stage stem cell maturation when expressed on CD34+bone marrow cells, and represents more mature hematopoietic progenitor cells. We observed that freshly isolated CD34+bone marrow cells express LFA-1, and that the level of expression is highly variable. Interestingly, the expression of the LFA-1 specific activation epitope L16 on these cells is low, even after culture. This demonstrates that LFA-1 is not activated, as was confirmed by low adhesion to ICAM-1. Culturing sorted CD34+LFA-1 +cells in single cell per well assays in medium supplemented with SCF, Epo, IL-3, IL-6, GM-CSF, and G-CSF revealed that they gave rise to dispersed macrophage-like colonies, supporting the notion that CD34+LFA-1+cells indeed consist of a mature committed cell population. In contrast, sorted CD34+LFA-1-cells had high proliferative potential and developed into large multilineage colonies within 14 days of culture. Unanticipated, in time course experiments we observed that these CD34+LFA-1-cells expressed LFA-1 within 24 hours upon culture. This induction was neither caused by the monoclonal antibody used to tag CD34 cells, nor dependent on growth factors present in the medium. These findings demonstrate that two populations of CD34+LFA-1+cells can be discriminated: leukocyte lineage committed CD34+ cells in freshly isolated bone marrow cells, and multipotent CD34+cells that acquired LFA-1 upon in vitro culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA-1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, providing those cells with adhesion receptors enabling migration in the periphery.

Previously, we have shown that interleukin (IL)-8 induces the rapid (15 to 30 minutes) mobilization of hematopoietic progenitor cells (HPC) in mice. Because integrins are essential for adhesion and transendothelial migration of HPC, we studied the involvement of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) in IL-8–induced mobilization. After a single injection of blocking anti–LFA-1 antibodies, no mobilization of colony-forming cells was observed. In addition, when mice were pretreated with anti–LFA-1 or saline and subsequently injected with 30 μg of IL-8, mobilization of HPC was completely blocked. We showed that this was not due to anti–LFA-1 antibodies affecting colony formation, as addition of anti–LFA-1 antibodies to colony cultures in semisolid medium had no inhibitory activity. Also, anti-intercellular adhesion molecule (ICAM)-1 antibodies, directed to the main ligand of LFA-1 significantly inhibited the IL-8–induced mobilization. Furthermore, IL-1–induced mobilization was significantly inhibited by anti–LFA-1 antibodies. Because LFA-1 is reported to be expressed on more differentiated HPC, it was considered that the IL-8–induced mobilization of more primitive HPC would not be blocked by anti–LFA-1 antibodies. Transplantation of blood-derived mononuclear cells (MNC) from IL-8–mobilized animals pretreated with anti–LFA-1 antibodies protected only 25% of lethally irradiated recipient mice, whereas the radioprotection rate of control mice transplanted with MNC derived from IL-8-mobilized animals was 86% (P< .01). Anti-LFA–1 antibodies did not interfere with stem cell homing, as transplantation of IL-8-mobilized blood MNC, incubated in vitro with these antibodies resulted in 100% radioprotection. We conclude that anti–LFA-1 antibodies completely prevent the rapid mobilization of colony-forming cells and of cells with radioprotective capacity induced by IL-8. These results indicate a major role for the β2-integrin LFA-1 in the IL-8–induced mobilization of hematopoietic stem cells.

ABSTRACTGlioblastoma multiforme (GBM) is the most aggressive brain tumor and median survival time with current therapies is only 14.6 mo. Although multiple immunotherapeutic strategies are being explored, efficacy remains poor. In order to improve immunotherapy for GBM, we propose to combine currently used endogenous with human cytomegalovirus (HCMV) specific antigens expressed on cancer cells.

In vivo dendritic-cell targeting constitutes a promising strategy for anticancer vaccination. Here, we discuss the usage of multivalent DC-SIGN-targeting glycan platforms that allow for the efficient routing of antigens to the endo-lysosomal pathway as well as to a yet uncharacterized cross-presentation mechanism inducing CD4+and CD8+T-cell responses.

Review: 137 refs.

Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell–specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galβ1-4GlcNAc, and binding to neoglycoconjugates containing only α-fucose or oligosaccharides with a terminal α1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.