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Metal hypersensitivity, a disorder of the immune system, typically manifests as contact hypersensitivity to metals during daily contact. The molecular mechanism whereby metals enter the body and cause allergic disorders remains elusive. It is thought that eluted metal ions are captured by dendritic cells (DCs) and transferred to the draining lymph nodes to activate T cells. Here, for the first time, we used a metal indicator Newport Green to locate the most common allergenic metal, nickel (Ni) ions, captured by DCs and transferred to lymph nodes. Capturing Ni ions did not affect the activity of DCs. Ni ions entered DCs and showed positive staining in keratinocytes. A time varied quantitative analysis demonstrated that at 1 h, a small amount of Ni ions was observed in the epidermal sheet. After 6 h, the number of Ni ions that entered the epidermal sheet reached a peak and remained constant for a few days and were gradually emitted 48 h later. In the cervical lymph nodes of mice, accumulation of Ni ions reached a peak within 24 h and then gradually decreased. The findings of this study will contribute to the development of effective diagnoses and treatment methods for patients allergic to Ni.

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.

Tuberculosis (TB) continues to be an important and unresolved medical problem. About a quarter of mankind is infected with Mycobacterium tuberculosis, and about 5–10% of these people eventually develop TB. Macrophages and CD4+ T cells are considered the key cells providing defense against TB infection. The role of neutrophils in TB is less well defined. Neutrophils are short-lived granulocytes among first migrate into the infectious lung tissue and phagocy tose mycobacteria. On the one hand, there is evidence for protective role of neutrophils in TB released via anti-microbial peptides inhibiting mycobacterial growth, up-regulation of CD4+ T-cell activation, and dendritic cell migration in the lymph nodes. On the other hand, infection of genetically TB susceptible animals leads to an overwhelming lung neutrophil inflammation, development of necrotic granulomata, and a rapid death. Neutrophils act directly or indirectly on mycobacteria by different oxidative or other reactions including neutrophil extracellular traps (NETs) formation. Phagocytosis of mycobacteria by neutrophils is accompanied by the production of pro-inflammatory factors, thus making neutrophils active participants of inflammation in all stages of the infectious process. Finally, neutrophils die by apoptosis or necrosis. Necrosis of neutrophils, which is activated by reactive oxygen species, also prolongs the inflammation. In this way, there is strong evidence that neutrophils are the cells involved in the transition of infection to the terminal stage, participating in lung tissue destruction. Although neutrophils evolutionary developed many ways to resist pathogens, it is likely, that neutrophils do not possess sufficient anti-mycobactericidal capacities due to the development of many adaptations allowing mycobacteria to survive inside the neutrophils. Neutrophils effectively phagocytose but poorly kill mycobacteria, thus hiding bacilli from more efficient killers, macrophages, and playing the role of the “Trojan Horse”. In this review, we summarize the data on the involvement of neutrophils in TB inflammation. We discuss their ambiguous role in pathogenesis which depends upon mycobacterial virulence, host genetics, dynamics of migration to inflammatory foci, and persistence during initial and chronic stages of the infectious process.

Dendritic cells (DCs) are the key link between innate immunity and adaptive immunity and play crucial roles in both the promotion of immune defense and the maintenance of immune tolerance. The trafficking of distinct DC subsets across lymphoid and nonlymphoid tissues is essential for DC-dependent activation and regulation of inflammation and immunity. DC chemotaxis and migration are triggered by interactions between chemokines and their receptors and regulated by multiple intracellular mechanisms, such as protein modification, epigenetic reprogramming, metabolic remodeling, and cytoskeletal rearrangement, in a tissue-specific manner. Dysregulation of DC migration may lead to abnormal positioning or activation of DCs, resulting in an imbalance of immune responses and even immune pathologies, including autoimmune responses, infectious diseases, allergic diseases and tumors. New strategies targeting the migration of distinct DC subsets are being explored for the treatment of inflammatory and infectious diseases and the development of novel DC-based vaccines. In this review, we will discuss the migratory routes and immunological consequences of distinct DC subsets, the molecular basis and regulatory mechanisms of migratory signaling in DCs, and the association of DC migration with the pathogenesis of autoimmune and infectious diseases.

Programmed Death-1 (PD-1)/Programmed Death-Ligand 1 (PD-L1) interactions are critical for dampening the immune response to both self and foreign antigens. The signaling of PD-L1 via its cytoplasmic domain, rather than through its interactions with PD-1 via the extracellular domain, has been termed PD-L1 reverse signaling. While this signaling is beneficial for cancer progression, little is understood about the consequences of PD-L1 reverse signaling in immune cells that express PD-L1 at steady state or in response to infection. Loss of PD-L1 during infection leads to unchecked T cell proliferation and increased autoimmune T cell responses. While the T cell intrinsic role of PD-1 for inhibiting T cell responses has been well explored, little to no effort has been directed at investigating the consequences of PD-L1 reverse signaling on the DCs interacting with PD-1+ T cells. We recently reported a defect in dendritic cell trafficking from the skin to the draining lymph node following immunization or infection in the absence of PD-L1. We demonstrated that a region within the cytoplasmic tail was responsible for the defect in dendritic cell trafficking. Here, we review the processes involved in dendritic cell trafficking and highlight what we know about PD-L1 expression, PD-L1 post-translational modifications, PD-L1 intracellular interactions and PD-L1 extracellular interactions.

Background: To explore the interlink between conjunctival goblet and corneal dendritic cell density after six months of lens wear and to predict dendritic cell migration to the central cornea based on goblet cell loss in the conjunctiva as a response to contact lens wear. Methods: Sixty-nine subjects who had never previously worn contact lenses were observed for six months; 46 were fitted with contact lenses and 21 served as a control group. Corneal confocal microscopy was used to quantify goblet and dendritic cell density before and after six months of daily lens wear. Symptomatic and asymptomatic groups were identified in the lens-wearing group using a combination of signs and symptoms present. Pearson's correlation was used to determine associations between the total change of cell densities after six months of lens wear. Results: At baseline, there was no association between conjunctival goblet and corneal dendritic cell density (p > 0.05). After six months, there was an inverse association between the absolute change of conjunctival goblet and corneal dendritic cell density (ρ = −0.34, p = 0.03) in all participants (n = 69). Dendritic cell density in the central cornea was increased by 1.5 cells/mm2 for every decrease of 1 goblet cell/mm2 in the conjunctiva. Conclusions: After six months of wear, contact lens-induced goblet cell loss can partially predict resident corneal dendritic cell migration to the central cornea (observed as an increase in dendritic cell density). The associations between total cell density change after six months was established in wearers regardless of lens symptomatology, suggesting that cell density changes as a physiological adaptation to regulate the effect of contact lens wear on the ocular surface.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells that act to bridge innate and adaptive immunity. DCs are critical in mounting effective immune responses to tissue damage, pathogens and cancer. Immature DCs continuously sample tissues and engulf antigens via endocytic pathways such as phagocytosis or macropinocytosis, which result in DC activation. Activated DCs undergo a maturation process by downregulating endocytosis and upregulating surface proteins controlling migration to lymphoid tissues where DC-mediated antigen presentation initiates adaptive immune responses. To traffic to lymphoid tissues, DCs must adapt their motility mechanisms to migrate within a wide variety of tissue types and cross barriers to enter lymphatics. All steps of DC migration involve cell–cell or cell–substrate interactions. This review discusses DC migration mechanisms in immunity and cancer with a focus on the role of cytoskeletal processes and cell surface proteins, including integrins, lectins and tetraspanins. Understanding the adapting molecular mechanisms controlling DC migration in immunity provides the basis for therapeutic interventions to dampen immune activation in autoimmunity, or to improve anti-tumour immune responses.

Purpose of review Dendritic cells are critical in directing inflammatory versus tolerogenic responses. As the burden of allergic disease rises worldwide, increased understanding of mechanisms underlying these diseases is needed. This review highlights research demonstrating how dendritic cells influence allergic disease development, providing important mechanistic insights into current clinical management strategies as well as potential areas of focus for future development of novel therapeutic strategies. Recent findings Recent studies continue to elucidate dendritic cell-associated pathways which can either promote or prevent allergic inflammation. Mechanisms involved include various aspects of dendritic cell activity, from antigen sampling and dendritic cell migration to complex dendritic cell interactions with other immune cells, infectious agents and allergens. A deeper understanding of these mechanisms and how dendritic cells promote tolerance provides insight into potential strategies to therapeutically target dendritic cells in the management of allergic disease. Summary Recent discoveries illustrate crucial roles of dendritic cells as regulators of inflammatory versus tolerant cascades. Building on lessons from oncologic strategies for harnessing dendritic cells to promote antitumor responses, several novel pathways could also be targeted to promote dendritic cell-mediated tolerogenesis in the context of allergy. Additional studies are needed to further define the roles and potential effects of dendritic cells in these potential strategies to reduce allergic inflammation.

Enteric-coated formulations using Eudragit® polymers have been extensively used for delivering drugs to the lower gastrointestinal tract. However, these drug-delivery systems cannot accurately deliver the therapeutic cargoes to colon because of early degradation of the polymers at alkaline pH of the small intestine. Here, we describe a precise method of delivering drugs to inflammation sites in colon using an oral drug delivery system. Tacrolimus (FK506)-loaded microspheres were prepared using a thioketal-based polymer that releases drug in response to reactive oxygen species (ROS), which are abundantly produced at the sites of inflammation in acute colitis. Orally-administered FK506-loaded thioketal microspheres (FK506-TKM) led to a substantial accumulation of FK506 in inflamed colon and effectively alleviated dextran-sulfate sodium (DSS)-induced murine colitis. At the molecular level, FK506-TKM significantly inhibited infiltration of CD4+ and CD8+ T lymphocytes in colon and differentiation of CD4+ T cells into Th1 and Th17 cells in colon-draining mesenteric lymph nodes via restricting dendritic cell migration from colon. Our findings indicate orally-administered thioketal-based drug delivery system as a promising means of treating acute inflammatory bowel diseases.

The coordination of cell migration of immune cells is a critical aspect of the immune response to pathogens. Dendritic cells (DCs), the sentinels of the immune system, are exposed to complex tissue microenvironments with a wide range of stiffnesses. Recent studies have revealed the importance of mechanical cues in immune cell trafficking in confined 3D environments. However, the mechanism by which stiffness modulates the intrinsic motility of immature DCs remains poorly understood. Here, immature DCs were found to navigate confined spaces in a rapid and persistent manner, surveying a wide range when covered with compliant gels mimicking soft tissues. However, the speed and persistence time of random motility were both decreased by confinement in gels with higher stiffness, mimicking skin or diseased, fibrotic tissue. The impact of stiffness of surrounding tissue is crucial because most in vitro studies to date have been based on cellular locomotion when confined by microfabricated polydimethylsiloxane structures. Our study provides evidence for a role for environmental mechanical stiffness in the surveillance strategy of immature DCs in tissues.

A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease.

Immunotherapies, such as dendritic cell- (DC-)based therapies, are useful for treating cancer as an alternative to or in combination with traditional therapies. Cells must migrate to lymphoid organs to be effective and the magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With less than 10% of cells expected to reach their destination, there is a need for an imaging modality capable of sensitively and quantitatively detecting cells. MRI has been used to track DC using iron and 19F methods, with limitations. Quantification of iron-induced signal loss is indirect and challenging; 19F signal is directly quantifiable but lacks sensitivity. Magnetic Particle Imaging (MPI) directly detects superparamagnetic iron oxide nanoparticles (SPIO) and enables quantitation of low numbers of SPIO-labeled cells. Here we describe the first study using MPI to track and quantify the migration of DC, injected into the footpads of C57BL/6 mice, to the popliteal lymph nodes (pLNs). As DC migrate from the site of injection to the lymph nodes, we measured a decrease in signal in the footpads and an increase in signal at the pLNs. The presence of SPIO-labeled DC in nodes was validated by ex vivo MPI and histology. By measuring the iron mass per cell in samples of labeled cells, we were able to provide an estimate of cell number for each source of signal and we report a sensitivity of approximately 4000 cells in vivo and 2000 cells ex vivo. For some mice, MPI was compared to cellular MRI. We also bring attention to the issue of resolving unequal signals within close proximity, a challenge for many pre-clinical studies using a highly concentrated tracer bolus that over shadows nearby lower signals. This study demonstrates the clear advantage of MPI to detect and quantify cells in vivo, bridging the gap left by cellular MRI, and all other in vivo imaging modalities, and opening the door for quantitative imaging of cellular immunotherapies.

Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MCOLN1), enabling the local activation of myosin II at the cell rear. Taken together, our findings identify Rab7b as the missing physical link between lysosomes and the actomyosin cytoskeleton, allowing control of immune cell migration through lysosomal signaling. This article has an associated First Person interview with the first author of the paper., Summary: The small GTPase Rab7b bridges the lysosomal Ca2+ channel TRPML1 to myosin II, thus enabling the local activation of myosin II at the cell rear and promoting fast migration of dendritic cells.

Lymphatic vessels are often considered passive conduits that rapidly flush antigenic material, pathogens, and cells to draining lymph nodes. Recent evidence, however, suggests that lymphatic vessels actively regulate diverse processes from antigen transport to leukocyte trafficking and dietary lipid absorption. Here we tested the hypothesis that dermal lymphatic transport is dynamic and contributes to innate host defense during viral infection. We demonstrate that cutaneous vaccinia virus infection activates the tightening of lymphatic interendothelial junctions, termed zippering, in a VEGFA/VEGFR2-dependent manner. Both antibody-mediated blockade of VEGFA/VEGFR2 and lymphatic-specific deletion of Vegfr2 impaired lymphatic capillary zippering and increased fluid flux out of tissue. Strikingly, inhibition of lymphatic zippering allows viral dissemination to draining lymph nodes independent of dendritic cell migration and impairs CD8+ T cell priming. These data indicate that infection-induced dermal lymphatic capillary zippering is a context-dependent, active mechanism of innate host defense that limits interstitial fluid and virion flux and promotes protective, anti-viral CD8+ T cell responses.SummaryCutaneous infection with vaccinia virus induces VEGFR2-dependent dermal lymphatic capillary zippering. This tightening of lymphatic junctions exacerbates tissue edema, sequesters virus, and promotes anti-viral CD8+ T cell responses. Dermal lymphatic capillaries are therefore an active component of innate host defense.

Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but failed in the mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is known for regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and immunoregulation are unclear. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockades. Finally, we identified that microRNA-552 negatively regulates ACKR4 expression in human CRC. Taken together, our work identifies a critical mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC tumors.LAY SUMMARYOur study demonstrated that Atypical Chemokine Receptor 4 (ACKR4) is downregulated in human colorectal cancer (CRC) tissues compared with normal colon tissues. Loss of ACKR4 in human CRC tissues is associated with a weak anti-tumor immune response. Knockdown of ACKR4 in tumor cells impairs the dendritic cell migration from the tumor tissue to the tumor-draining lymph nodes (TdLNs), causing inadequate tumor-specific T-cell expanding and insensitivity to immune checkpoint blockades. However, loss of ACKR4 in tumor stromal cells does not significantly affect anti-tumor immunity. In human CRC tumors, high expression of microRNA-552 is a mechanism leading to ACKR4 downregulation. Our study revealed a novel mechanism that leads to the poor immune response in a subset of CRC tumors and will contribute to the framework for identifying new therapies against this deadly tumor.

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-β treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Our previous study demonstrated an indispensable role of Peyer’s patches (PPs) for the induction of antigen-specific secretory (S)IgA antibody responses after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella-Tox C). In this study, we defined the PP lymphoid structures and immune cells required for the induction of mucosal SIgA antibody responses. Adoptive transfer of mononuclear cells (MNCs) from PPs into PP-deficient (PP-null) mice failed to elicit tetanus toxoid (TT)-specific mucosal immunity. However, when the same PP MNCs were transferred into lethally irradiated PP-normal recipient mice, PP MNCs preferentially emigrated to recipient PPs, leading to PP lymphoid structures and TT-specific SIgA antibody responses. Significantly reduced numbers of TT-specific IgA antibody-forming cells were detected in the mesenteric lymph nodes (MLNs) and intestinal lamina propria of mice when surface expression of the sphingosine 1-phosphate receptor on lymphocytes was inhibited by its agonist FTY720. However, FTY720 treatment did not alter dendritic cell migration or Salmonella dissemination into these tissues. When rSalmonella-Tox C-stimulated CD4+ T cells isolated from PPs, MLNs and the spleen were co-cultured with B cells from these tissues, significantly increased levels of TT-specific IgA antibody responses were exclusively induced in cultures containing PP B cells. Furthermore, surface IgA+ PP B cells produced TT-specific IgA antibody responses in vitro. These findings suggest that PP lymphoid structures and surface IgA+ PP B cells are essential elements for the induction of antigen-specific intestinal SIgA antibody responses to oral Salmonella.

Type 2 immunity in the lung is promoted through the release of innate cytokines, including TSLP, from lung structural cells. These cytokines drive Type 2 immunity in part through upregulation of OX40L on dendritic cells (DCs). DCs expressing OX40L are potent inducers of Th2 differentiation. We have shown previously that STAT6 inhibitory peptide (STAT6-IP), a cell penetrating peptide designed to inhibit the STAT6 transcription factor, reduces the induction of Th2 adaptive immunity in murine models of respiratory syncytial virus infection. Here we show that intranasal administration of STAT6-IP at the time of antigen priming with ovalbumin (OVA), in conjunction with the Nod2 agonist, MDP, reduced frequencies of CD11b+ lung DCs expressing OX40L. Consistent with these reductions, fewer activated DCs were localized to the lung draining lymph nodes in STAT6-IP-treated mice. Upon OVA challenge four weeks later, mice treated with STAT6-IP at the time of OVA/MDP priming did not develop airway hyperresponsiveness (AHR) and had reduced influx of eosinophils into the airways, mucus production, and serum OVA-specific IgE levels. Our findings provide evidence that the long-lasting inhibitory effects of STAT6-IP are due in part to inhibition of DC responses that drive maladaptive Th2 adaptive immunity and allergic airways disease.

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

Summary Immunogenic tumor cell death enhances anti-tumor immunity. However, the mechanisms underlying this effect are incompletely understood. We established a system to induce tumor cell death in situ and investigated its effect on dendritic cell (DC) migration and T cell responses using intravital photolabeling in mice expressing KikGR photoconvertible protein. We demonstrate that tumor cell death induces phagocytosis of tumor cells by tumor-infiltrating (Ti)-DCs, and HMGB1-TLR4 and ATP-P2X7 receptor signaling-dependent Ti-DC emigration to draining lymph nodes (dLNs). This led to an increase in anti-tumor CD8+ T cells of memory precursor effector phenotype and secondary tumor growth inhibition in a CD103+ DC-dependent manner. However, combining tumor cell death induction with lipopolysaccharide treatment stimulated Ti-DC maturation and emigration to dLNs but did not improve tumor immunity. Thus, immunogenic tumor cell death enhances tumor immunity by increasing Ti-DC migration to dLNs where they promote anti-tumor T cell responses and tumor growth inhibition.

Signaling by the HGF receptor/Met in skin-resident Langerhans cells (LC) and dermal dendritic cells (dDC) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. By employing a conditional Met-deficient mouse model (Metflox/flox) we addressed the role of Met-signaling in distinct steps of LC/dDC emigration from skin. Met deficiency did not prevent the decline in expression of the adhesion molecules E-cadherin and EpCAM in LC upon activation and consequently detachment from the surrounding tissue and migration towards dermis. However, Met-deficient LC failed to efficiently cross the extracellular matrix (ECM) rich basement membrane between epidermis and dermis. We found that Met deficiency severely impaired podosome formation in DC and concomitantly decreased proteolytic degradation of gelatin. We further observed that Met-signaling by HGF reduced adhesion of bone marrow-derived LC to various ECM factors and enhanced the motility of Met-signaling competent DC in a 3D collagen matrix, which was not the case for Met-deficient LC/DC. In contrast, we found no impact of Met-signaling on the integrin-independent amoeboid migration of DC in response to the CCR7 chemokine CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.

Antigen cross-presentation to cytotoxic CD8+ T cells is crucial for the induction of anti-tumor and anti-viral immune responses. Recently, co-encapsulation of photosensitizers and antigens into microspheres and subsequent photochemical internalization (PCI) of antigens in antigen presenting cells has emerged as a promising new strategy for inducing antigen-specific CD8+ T cell responses in vitro and in vivo. However, the exact cellular mechanisms have hardly been investigated in vivo, i.e., which cell types take up antigen-loaded microspheres at the site of injection, or in which secondary lymphoid organ does T cell priming occur? We used spray-dried poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with ovalbumin and the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) to investigate these processes in vivo. Intravital microscopy and flow cytometric analysis of the murine ear skin revealed that dendritic cells (DCs) take up PLGA microspheres in peripheral tissues. Illumination then caused photoactivation of TPCS2a and induced local tissue inflammation that enhanced CCR7-dependent migration of microsphere-containing DCs to tissue-draining lymph nodes (LNs), i.e., the site of CD8+ T cell priming. The results contribute to a better understanding of the functional mechanism of PCI-mediated vaccination and highlight the importance of an active transport of vaccine microspheres by antigen presenting cells to draining LNs.

This paper outlines changes to the ocular surface caused by contact lenses and their degree of clinical significance. Substantial research and development to improve oxygen permeability of rigid and soft contact lenses has meant that in many countries the issues caused by hypoxia to the ocular surface have largely been negated. The ability of contact lenses to change the axial growth characteristics of the globe is being utilised to help reduce the myopia pandemic and several studies and meta-analyses have shown that wearing orthokeratology lenses or soft multifocal contact lenses can reduce axial length growth (and hence myopia).However, effects on blinking, ptosis, the function of Meibomian glands, fluorescein and lissamine green staining of the conjunctiva and cornea, production of lid-parallel conjunctival folds and lid wiper epitheliopathy have received less research attention. Contact lens wear produces a subclinical inflammatory response manifested by increases in the number of dendritiform cells in the conjunctiva, cornea and limbus. Papillary conjunctivitis is also a complication of all types of contact lenses. Changes to wear schedule (daily disposable from overnight wear) or lens materials (hydrogel from SiHy) can reduce papillary conjunctivitis, but the effect of such changes on dendritic cell migration needs further study. These changes may be associated with decreased comfort but confirmatory studies are needed. Contact lenses can affect the sensitivity of the ocular surface to mechanical stimulation, but whether these changes affect comfort requires further investigation.In conclusion, there have been changes to lens materials, design and wear schedules over the past 20+ years that have improved their safety and seen the development of lenses that can reduce the myopia development. However, several changes to the ocular surface still occur and warrant further research effort in order to optimise the lens wearing experience.

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs thereby orchestrating adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and migrate to lymphoid organs to metastasize. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. To this, we established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6Dy649P1 and CCL21-S6Dy649P1 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit β-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6Dy649P1 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations.

Background Dendritic cells (DCs) play a key role in shaping T cell responses. To do this, DCs must be able to migrate to the site of the infection and the lymph nodes to prime T cells and initiate the appropriate immune response. Integrins such as β2 integrin play a key role in leukocyte adhesion, migration, and cell activation. However, the role of β2 integrin in DC migration and function in the context of infection-induced inflammation in the gut is not well understood. This study looked at the role of β2 integrin in DC migration and function during infection with the nematode worm Trichuris muris. Itgb2tm1Bay mice lacking functional β2 integrin and WT littermate controls were infected with T. muris and the response to infection and kinetics of the DC response was assessed. Results In infection, the lack of functional β2 integrin significantly reduced DC migration to the site of infection but not the lymph nodes. The lack of functional β2 integrin did not negatively impact T cell activation in response to T. muris infection. Conclusions This data suggests that β2 integrins are important in DC recruitment to the infection site potentially impacting the initiation of innate immunity but is dispensible for DC migration to lymph nodes and T cell priming in the context of T. muris infection.

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.