Regulation Of Dendritic Cell Migration Research Articles

CCR7+ dendritic cells sorted by binding of CCL19 show enhanced Ag-presenting capacity and antitumor potency.

Dendritic cell therapy has been a promising addition to the current armory of therapeutic options in cancer for more than 20 years but has not yet achieved breakthrough success. To successfully initiate immunity, dendritic cells have to enter the lymph nodes. However, experience to date of therapeutic dendritic cell administration indicates that this is frequently an extremely inefficient process. The major regulator of dendritic cell migration to the lymph nodes is the chemokine receptor CCR7 and in vitro generated dendritic cells typically display heterogeneous expression of this receptor. Here we demonstrate that positive selection for the dendritic cell subpopulation expressing CCR7, using a chemically-synthesized ligand:CCL19, enriches for cells with enhanced lymph node migration and Ag presentation competence as well as a chemokine expression profile indicative of improved interactions with T cells. This enhanced lymph node homing capacity of enriched CCR7+ cells is seen in comparison to a population of unsorted dendritic cells containing an equivalent number of CCR7+ dendritic cells. Importantly, this indicates that separating the CCR7+ dendritic cells from the CCR7- cells, rather than simple CCL19 exposure, is required to affect the enhanced lymph node migration of the CCR7+ cells. In models of both subcutaneous and metastatic melanoma, we demonstrate that the dendritic cells sorted for CCR7 expression trigger enhanced CD8 T-cell driven antitumor immune responses which correlate with reduced tumor burden and increased survival. Finally, we demonstrate that this approach is directly translatable to human dendritic cell therapy using the same reagents coupled with clinical-grade flow-cytometric sorting.

The transcription factor NR4A3 controls CD103+ dendritic cell migration.

The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.

Generation of potent dendritic cells with improved migration ability through p-cofilin and sarco/endoplasmic reticulum Ca2+ transport ATPase 2 regulation

Background aimsIt is important to improve the migratory ability of dendritic cells (DCs) and to increase DC potency for successful DC-based cancer immunotherapy. The intracellular Ca2+ signaling pathway has an important role on the regulation of DC migration. Our preliminary studies revealed that sarco/endoplasmic reticulum Ca2+ transport ATPase 2 (SERCA2) expression was inversely related to DC migratory capacity, and the expression level of p-cofilin and SERCA2 on mature DCs showed a counter-trend. MethodsWe selected the appropriate six maturation cocktails on the basis of the expression levels of SERCA2 and p-cofilin and investigated the functional characteristics and migratory capacity of mature DCs. Among the these six maturation cocktails, DCIFN-γ/IL-1β/Poly-I:C showed potent type 1 immune response with interleukin (IL)-12p70 production and strong Th1-polarization, and this DC elicited strong antigen-specific cytotoxic T-lymphocyte responses. ResultsInterestingly, DCIFN-γ/IL-1β/Poly-I:C showed lower expression of SERCA2 and higher expression of p-cofilin compared with those matured with the use of other cocktails. In vitro migration assay showed that DCs matured with the use of this maturation cocktail had significantly increased migratory ability compared with αDC1s and other DCs. ConclusionsInterferon-γ, IL-1β and Poly-I:C maturation cocktail may be used in the field of cancer immunotherapy to generate potent immune-stimulatory DCs with improved type 1 immune response and migration capacity.

Regulator of dendritic cell migration, ASAP1 is associated with increased susceptibility to tuberculosis.

Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration Curtis J et al. (2015) Nature Genetics 47(5):523-527.

Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration.

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 pulmonary TB patients and 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10−11 for rs4733781; P = 1.0 × 10−10 for rs10956514). Dendritic cells (DCs) showed high level of ASAP1 expression, which was reduced after M. tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential novel mechanism that predisposes to TB.

Business as usual: the p35 isoform of human CD74 retains function in antigen presentation

Business as usual: the p35 isoform of human CD74 retains function in antigen presentation

The HGF Receptor/Met Tyrosine Kinase Is a Key Regulator of Dendritic Cell Migration in Skin Immunity

The Met tyrosine kinase has a pivotal role in embryonic development and tissue regeneration, and deregulated Met signaling contributes to tumorigenesis. After binding of its cognate ligand hepatocyte growth factor, Met signaling confers mitogenic, morphogenic, and motogenic activity to various cells. Met expression in the hematopoietic compartment is limited to progenitor cells and their Ag-presenting progeny, including dendritic cells (DCs). In this study, we demonstrate that Met signaling in skin-resident DCs is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. By using a conditional Met-deficient mouse model (Met(flox/flox)), we show that Met acts on the initial step of DC release from skin tissue. Met-deficient DCs fail to reach skin-draining lymph nodes upon activation while exhibiting an activated phenotype. Contact hypersensitivity reactions in response to various contact allergens is strongly impaired in Met-deficient mice. Inhibition of Met signaling by single-dose epicutaneous administration of the Met kinase-specific inhibitor SU11274 also suppressed contact hypersensitivity in wild-type mice. Additionally, we found that Met signaling regulates matrix metalloproteinase MMP2 and MMP9 activity, which is important for DC migration through extracellular matrix. These data unveil Met signaling in DCs as a critical determinant for the maintenance of normal immune function and suggest Met as a potential target for treatment of autoimmune skin diseases.

Four-and-a-Half LIM Domain Protein 2 Is a Novel Regulator of Sphingosine 1-Phosphate Receptor 1 in CCL19-Induced Dendritic Cell Migration

We identified the four-and-a-half LIM domain protein 2 (FHL2) as a novel regulator of CCL19-induced dendritic cell (DC) migration. Initiation of migration is a hallmark of DC function and plays a central role in the induction and regulation of immune responses. In vivo, DCs continuously acquire Ag in the periphery and migrate to draining lymph nodes, under the influence of local environmental chemotactic factors like CCL19/21 or sphingosine 1-phosphate (S1P). We investigated the role of S1P- and RhoA-regulated FHL2 in this process. We found reduced nuclear localization of FHL2 in mature bone marrow-derived DCs (BMDCs), compared with immature BMDCs, following stimulation with CCL19. Furthermore, in vitro-generated murine FHL2(-/-) BMDCs displayed a significantly increased migratory speed, directionality, and migratory persistence toward the chemokine CCL19 compared with wild-type BMDCs. Moreover, in vivo, FHL2(-/-) BMDCs showed increased migration toward lymphoid organs. FHL2(-/-) BMDCs increased the expression of S1PR1, which was associated with greater Rac activation. An S1PR1 antagonist and knock-down of S1PR1 abrogated the increased migratory speed of FHL2(-/-) BMDCs. Our results identify FHL2 as an important novel regulator of DC migration via regulation of their sensitivity toward environmental migratory cues like S1P and CCL19.

Dermal Fibroblasts Promote the Migration of Dendritic Cells

Migration of dendritic cells (DCs) from skin to lymph nodes on activation is an essential step in the initiation of an adequate immune response. The dermal microenvironment including stromal cells and their soluble factors might be involved in the regulation of DC migration. To focus on the role of dermal fibroblasts, we studied whether interaction of DCs with fibroblasts promotes the migration of DCs. DCs were co-cultured with resting fibroblasts or with tumor necrosis factor (TNF)alpha/IL-1beta-activated fibroblasts to mimic an inflammatory microenvironment. Interaction of DCs with TNFalpha/IL-1beta-stimulated fibroblasts increased the secretion of matrix metalloproteinase-9 (MMP-9) from DCs within 6 hours compared with DCs alone or DCs stimulated with lipopolysaccharide or TNFalpha/IL-1beta. In contrast, unstimulated fibroblasts did not affect MMP-9 secretion. IL-6 released by TNFalpha/IL-1beta-stimulated fibroblasts was identified as a factor responsible for fibroblast-stimulated MMP-9 secretion from DCs. In accordance with the elevated MMP-9 release, on co-culture with TNFalpha/IL-1beta-stimulated fibroblasts, DCs migrated significantly more effectively through matrigel matrices than did TNFalpha/IL-1beta-stimulated DCs. This was inhibited by a selective blocking of MMP-9, indicating the importance of MMP-9 for this migratory capacity of DCs. In summary, fibroblasts in the local dermal microenvironment are capable of potentiating the migratory capacity of DCs, and thus have the potential to actively participate in the regulation of a cutaneous immune response.

Regulation of Dendritic Cell Migration by CD74, the MHC Class II-Associated Invariant Chain

Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur independently. We found that the major histocompatibility complex II-associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.

PGE2-induced metalloproteinase-9 is essential for dendritic cell migration

Following antigen acquisition and maturation, dendritic cells (DCs) disengage from the extracellular matrix, cross basement membranes, and travel to draining lymph nodes to activate T cells. CCR7 expression is necessary but not sufficient for the directional migration of DCs. Prostaglandin E2 (PGE2), present in inflammatory sites, induces DC migration, presumably by enacting a migration-permissive gene expression program. Since regulation of DC migration is highly important for their use in vaccination and therapy, we examined the PGE2-induced changes in the expression of metalloproteinases (MMPs). Our results indicate that PGE2 significantly up-regulates MMP-9 expression, induces both secreted and membrane-bound MMP-9, and that in turn, DC-derived MMP-9 is essential for DC chemotaxis in response to the CCR7 ligand CCL19, Matrigel migration, and in vivo migration in both wild-type and MMP-9-deficient hosts. We conclude that DCs matured within inflammatory sites require both CCR7 and PGE2-induced MMP-9 for their directional migration to draining lymph nodes.

Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function

AbstractTrafficking of dendritic cells (DCs) to peripheral tissues and to secondary lymphoid organs depends on chemokines and lipid mediators. Here, we show that bone marrow–derived DCs (BM-DCs) express functional leukotriene B4 (LTB4) receptors as observed in dose-dependent chemotaxis and calcium mobilization responses. LTB4, at low concentrations, promoted the migration of immature and mature DCs to CCL19 and CCL21, which was associated with a rapid (30-minute) increase of CCR7 expression at the membrane level. At longer incubation times (6 hours), gene array analysis revealed a promoting role of LTB4, showing a significant increase of CCR7 and CCL19 mRNA levels. BM-DCs cultured from BLT1−/− or BLT1/2−/− mice showed a normal phenotype, but in vivo BLT1/2−/−DCs showed dramatic decrease in migration to the draining lymph nodes relative to wild-type (WT) DCs. Consistent with these observations, BLT1/2−/− mice showed a reduced response in a model of 2,4-dinitro-fluorobenzene (DNFB)–induced contact hypersensitivity. Adoptive transfer of 2,4-dinitrobenzene sulfonic acid (DNBS)–pulsed DCs directly implicated the defect in DC migration to lymph node with the defect in contact hypersensitivity. These results provide strong evidence for a role of LTB4 in regulating DC migration and the induction of adaptive immune responses.

Suppression of the Bacterial Antigen‐Specific T Cell Response and the Dendritic Cell Migration to the Lymph Nodes by Osteopontin

Osteopontin (OPN) has been reported to enhance the interferon (IFN)-gamma-producing Th1-type T cell response through the induction of interleukin (IL)-12 and the suppression of IL-10. We therefore investigated whether OPN could enhance Th1 induction by vaccination against bacterial antigen in vivo. Unexpectedly, the co-inoculation of OPN suppressed the induction of IFN-gamma-producing CD4(+) T cells and T cell proliferative response after the subcutaneous heat-killed Listeria monocytogenes(HKLM) immunization. These results suggest that OPN down-regulates T cell priming. Since dendritic cells (DC) play a pivotal role in T cell priming, we next analyzed the effects of OPN on DC. The addition of OPN into the culture of either bone marrow-derived immature DC or an immature DC line JAWSII showed no effects on the expression of MHC class II, CD80, and CD86 molecules before and after HKLM stimulation. Consistently, in vitro OPN-treated DC showed a normal antigen-presenting function to an established Listeria-specific Th1-type T cells. However, when the DC were transferred into the footpad with HKLM and OPN, the migration of the transferred DC into the regional LN was suppressed in comparison to the DC transferred with HKLM alone. Furthermore, the addition of OPN into the culture of the DC line and HKLM severely suppressed the HKLM-induced expression of CCR7 chemokine receptor which is an important factor in the migration of DC into LN. All the results suggest the existence of an OPN-mediated negative feedback mechanism in the T cell immune response through the regulation of DC migration.

Novel Function of IFN-γ: Negative Regulation of Dendritic Cell Migration and T Cell Priming

IFN-gamma is considered to be a Th1 cytokine with immunomodulatory effects on a variety of immune cells. In this study, we determined whether dendritic cell (DC) function was aberrant in IFN-gamma knockout (GKO) mice. The results demonstrated that IFN-gamma deficiency did not interfere with bone marrow-derived DC development and maturation in vitro. However, functional analysis showed that bone marrow-derived DC from GKO mice had altered cytokine secretion, allostimulatory and Ag presentation capacity, chemokine receptor expression, and in vitro chemotaxis. LPS induced the recruitment of DC from different organs into the spleen; epicutaneously sensitized DC with hapten (FITC) accumulated in the draining lymph nodes and CD11c(+) DC levels in the draining lymph nodes from autoantigen (interphotoreceptor retinoid-binding protein) immunized mice were enhanced in GKO mice as compared with wild-type mice. After treatment of GKO mice with i.p. IFN-gamma injection restored IFN-gamma levels in vivo, DC migration decreased in response to LPS or FITC. IFN-gamma altered the adaptive immune responses in vivo, since T cell priming and IL-2 production were increased in interphotoreceptor retinoid-binding protein-immunized GKO mice. Furthermore, in IFN-gamma-treated GKO mice, experimental autoimmune uveitis score enhancement and T cell activation were eliminated. Taken together, IFN-gamma appears to play a negative regulatory role on in vivo DC function, resulting in suppression of Ag-specific T cell priming.

Nitric oxide and cGMP protein kinase (cGK) regulate dendritic-cell migration toward the lymph-node–directing chemokine CCL19

Dendritic-cell (DC) migration to secondary lymphoid organs is crucial for the initiation of adaptive immune responses. Although LPS up-regulates CCR7 on DCs, a second signal is required to enable them to migrate toward the chemokine CCL19 (MIP-3beta). We found that the nitric oxide (NO) donor NOR4 provides a signal allowing LPS-stimulated DCs to migrate toward CCL19. NO affects DC migration through both the initial activation of the cGMP/cGMP kinase (cGMP/cGK) pathway and a long-term effect that reduced cGK activity via negative feedback. Indeed, migration of DCs toward CCL19, unlike migration toward CXCL12 (SDF-1alpha), required inhibition of cGK. LPS increased both cGK expression and cGK activity as measured by phosphorylation of the key cGK target vasodilator-stimulated phosphoprotein (VASP). Because cGK phosphorylation of VASP can disrupt focal adhesions and inhibit cell migration, LPS-induced VASP phosphorylation may prevent DCs from migrating without a second signal. Long-term NOR4 treatment inhibited the increase in cGK-dependent VASP phosphorylation, releasing this brake so that DCs can migrate. NO has been implicated in the regulation of autoimmunity through its effect on T cells. Our results suggest that NO regulation of DC migration and cytokine production may contribute to the protective effects of NO in autoimmune disorders.

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming.

Antigen-pulsed dendritic cells (DCs) are used as natural adjuvants for vaccination, but the factors that influence the efficacy of this treatment are poorly understood. We investigated the parameters that affect the migration of subcutaneously injected mouse-mature DCs to the draining lymph node. We found that the efficiency of DC migration varied with the number of injected DCs and that CCR7+/+ DCs migrating to the draining lymph node, but not CCR7−/− DCs that failed to do so, efficiently induced a rapid increase in lymph node cellularity, which was observed before the onset of T cell proliferation. We also report that DC migration could be increased up to 10-fold by preinjection of inflammatory cytokines that increased the expression of the CCR7 ligand CCL21 in lymphatic endothelial cells. The magnitude and quality of CD4+ T cell response was proportional to the number of antigen-carrying DCs that reached the lymph node and could be boosted up to 40-fold by preinjection of tumor necrosis factor that conditioned the tissue for increased DC migration. These results indicate that DC number and tissue inflammation are critical parameters for DC-based vaccination.

Migration of dendritic cells into the lymphatics: the Langerhans cell example

Dendritic cells, including Langerhans cells of the epidermis and the mucous membranes, are key leukocytes for the initiation of adaptive immune responses as well as for the maintenance of peripheral tolerance. In the former regard they may well play an important, but as yet unrecognized role in the pathogenesis of Behcet's disease. Epidermal Langerhans cells may serve as a paradigm for their counterparts in the mucosae. These cells efficiently take up (microbial) antigens, they process them into immunogenic MHC–peptide complexes, and they transport this form of antigen to the lymph nodes via lymphatic vessels. Depending on the milieu where Langerhans cells have encountered antigen (inflammatory versus non-inflammatory/steady-state), they make T cells proliferate and acquire effector functions (immunity) or render them unresponsive or even delete them (tolerance), respectively. In addition, plasmacytoid dendritic cells, a recently characterized type of dendritic cells, may also directly trigger innate responses (e.g. by secretion of type I interferons in response to virus). Studying the pathways and the regulation of dendritic cell migration might help to unravel a possible involvement of dendritic cells in Behcet's disease. We present observations on the physical obstacles that dendritic cells migrating in the skin have to overcome until they reach dermal lymphatic vessels. Furthermore, we show that migration is critically dependent on the function of matrix metalloproteinases, in particular MMP-2 and MMP-9. It becomes evident that Langerhans cells indeed carry antigens (including self antigens such as melanosomes or apoptotoic bodies or tumor antigens such as particular cytokeratins) through the lymphatics. Given these observations it may be worth studying Langerhans cells and dermal dendritic cells in Behcet's disease.

A Close-Up View of Migrating Langerhans Cells in the Skin

Langerhans cells of the epidermis and dermal dendritic cells screen the skin for invading antigens. They initiate primary immune responses after migrating from sites of antigen uptake to lymphoid organs. The skin is a feasible model to study the morphology and regulation of dendritic cell migration. We therefore used murine skin explant cultures for tracking the pathways of dendritic cell migration by electron microscopy. Several novel observations are reported. (i) In 48 h cultures of epidermal sheets numerous Langerhans cells migrated out between keratinocytes extending long and thin cytoplasmic processes ("veils"). (ii) Langerhans cells in transition from epidermis to dermis were observed by transmission electron microscopy. Where Langerhans cells penetrated the basement membrane, the lamina densa was focally absent. (iii) This was highlighted by scanning electron microscopy, which presented the basement membrane as a tightly packed and dense network of fibrils. (iv) Scanning electron microscopy of the dermis revealed dendritic cells extending their cytoplasmic processes and clinging to collagen fibrils. (v) Entry of dendritic cells into dermal lymphatics was observed by transmission electron microscopy. It occurred by transmigration through intercellular spaces of adjacent endothelial cells. Entry through wide gaps between endothelial cells also seemed to take place. (vi) Dendritic cells inside the afferent lymphatics frequently carried material such as melanosomes and apoptotic bodies. These observations visualize the cumbersome pathway that dendritic cells have to take when they generate immunity.

Neuropeptides and the Immune System: Focus on Dendritic Cells

Neuropeptides are small, biologically active peptides derived from the central and peripheral nervous system. It is increasingly clear that besides their function as neurotransmitters, these peptides have an influence on almost all body functions including the immune system. Since dendritic cells are the most efficient antigen-presenting cells that stimulate naive T cells, thus promoting adaptive immunity, it is not surprising that interactions between neuropeptides and dendritic cells take place. The current review addresses several aspects of dendritic cell-related neuroimmunology and focuses on the role of neuropeptides as immunomodulators. Moreover, we present a novel concept of neuropeptide-mediated regulation of dendritic cell migration. The importance of chemokines in immunity is generally accepted. It may be that not enough attention has been paid to the possible role of nervous system-derived peptides in regulating immune reactions.