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Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications., From the Clinical Editor: The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Dendritic cells (DCs) are the most important professional antigen presenting cells that play a key role in initiating adaptive immune responses. The depletion and dysfunction of DCs contribute to the development of immunodeficiency or immunoparalysis in some lung diseases. In the present study, we investigated the effects of Fms-like tyrosine kinase-3 ligand (Flt3L) administration in vivo on lung DCs expansion to provide an experimental basis of Flt3L used as a potential therapeutic agent for the related lung disorders., Methods: Balb/c mice were randomly divided into Flt3L group (n = 10) and control group (n = 10). Each mouse in the Flt3L group received subcutaneous administration of Flt3L at a dose of 10 µg once daily for nine consecutive days. Lung histology was observed, and CD11c and CD205 were immunologically labeled in lung tissue sections. Low-density lung cells were separated by density gradient centrifugation, and then subsets and MHC-II/I-A(d) expression of DCs were analyzed by flow cytometry., Results: In the Flt3L group the number and density of DC-like cells were markedly increased compared with the control group, mainly distributed in the alveolar septa. Immunological labeling in situ found that there were significantly higher numbers of CD11c(+) and CD205(+) DCs in lung mesenchymal tissue (P < 0.05), where they formed a denser reticular formation. Flow cytometry analysis demonstrated that the proportions of myeloid CD11c(+)CD11b(+) DCs and plasmacytoid CD11c(+)CD45R/B220(+) DCs in the low-density lung cells in the Flt3L group were significantly higher compared with the control group; showing 3.17- and 3.3-fold increase respectively (P < 0.05). The proportion of CD11c(+) DCs expressing MHC-II/I-A(d+) was significantly increased, with a 2.7-fold increase as compared with the control group (P < 0.05)., Conclusions: Flt3L administration in vivo induces lung DCs expansion, favoring myeloid and plasmacytoid DC subsets, which are phenotypically more mature. Flt3L may be useful in the therapy to augment immune function of the lung.

The architectonics and cell composition of the human large arteries are not sufficiently understood. The present study is the first to undertake an analysis of the distribution and quantities of HLA-DR-expressing cells in grossly undiseased human intima using immunohistochemical and immunofluorescent analysis, complemented by the advantages of confocal microscopy. The study revealed a widespread distribution of HLA-DR-expressing cells throughout the intimal space where the cells were integrated into continuous networks via long cell processes. Numbers of HLA-DR+ cells were found to be significantly larger in the middle third of the intima than in the superficial and deep intimal portions. We speculate that a widespread distribution of HLA-DR-expressing cells in the intima of normal human aorta might play a role in the surveillance and maintenance of vascular homeostasis., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Purpose: The authors present a protocol for the in vivo evaluation, using different imaging techniques, of lymph node (LN) homing of tumor-specific dendritic cells (DCs) in a murine breast cancer model., Procedures: Bone marrow DCs were labeled with paramagnetic nanoparticles (MNPs) or (111)In-oxine. Antigen loading was performed using tumor lysate. Mature DCs were injected into the footpads of transgenic tumor-bearing mice (MMTV-Ras) and DC migration was tracked by magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT). Ex vivo analyses were performed to validate the imaging data., Results: DC labeling, both with MNPs and with (111)In-oxine, did not affect DC phenotype or functionality. MRI and SPECT allowed the detection of iron and (111)In in both axillary and popliteal LNs. Immunohistochemistry and γ-counting revealed the presence of DCs in LNs., Conclusions: MRI and SPECT imaging, by allowing in vivo dynamic monitoring of DC migration, could further the development and optimization of efficient anti-cancer vaccines.

The aim of this study is to investigate phenotypic and functional modulation of murine dendritic cells (DCs) with use of purified Glycyrrhizin (GL). These impacts of GL on DCs both from bone marrow derived DCs and established DC cell 2.4 were assessed with conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that the purified GL induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD80, CD83 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs2.4 cells were down- regulated after treatment with GL (which occurs when phagocytosis of DCs2.4 cells were decreased). Finally, we proved that GL increased the production of IL-12, IL-10 and decreased the production of tumor necrosis factor alpha (TNF-α). These data indicated that GL could promote maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that GL could exert positive modulation on murine DCs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Three dimensional (3D) electron microscopy techniques have become valuable tools for investigating cellular architecture and the processes that govern it. A vast amount of information is available in every 3D tomogram but the options for presenting this information in a clear and visually appealing way are limited. To address this, we developed D-CAT; a MatLab-application to accurately visualize the distribution of membrane proteins and/or membrane-bound structures. Presence (density) and distribution (clustering, depletion) are presented as color-coded areas on membranes. By using IMOD models both as input and output format, we ensure that the application fits within workflows common in the field of 3D electron microscopy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Indeterminate cell tumor is an extremely rare neoplasm that mainly occurs in the skin. We report a case of indeterminate cell tumor arising from the spleen, a previously unreported site for indeterminate cell tumor. Histologically, the tumor showed nests, nodules, and sheets of large polygonal cells with mostly oval nuclei; open chromatin; variable nucleoli; and abundant, eosinophilic cytoplasm. Some cells possessed irregularly convoluted nuclei with nuclear grooves and granular cytoplasm, suggestive of Langerhans cells. Immunohistochemically, the tumor cells were diffusely positive for S-100 and CD1a and negative for Langerin. No Birbeck granules were found by electron microscopy. Clinical and radiologic examination showed no other organomegaly or lymphadenopathy. A diagnosis of primary indeterminate cell tumor of the spleen was rendered. To the best of our knowledge, this is the first indeterminate cell tumor reported in the spleen. Biologic insights into dendritic cells in the spleen and the pertinent literature on these entities are reviewed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Lineage negative dendritic cells, or natural interferon-producing cells (NIPC), also referred to as plasmacytoid dendritic cells (pDC) constitute a small population of leukocytes secreting high levels of type I interferon (IFNα/β) in response to certain danger signals. Here, we provide initial data towards the identification of so far uncharacterised circulating bovine pDC like cells. A lineage negative cell population (LIN(-) cells) was isolated from PBMC which showed characteristics similar to that of pDC in other species. Isolated LIN(-) cells presented lymphoid morphology with a semi-crescent nucleus, extensive ER and Golgi network; indicative of pDC. In addition phenotypic analysis of LIN(-) cells described them as distinct from other bovine DC subsets; expressing both lymphoid and myeloid surface markers. LIN(-) cells did not express lineage specific markers, but were MHC class II(+), CD45RO(+), CD80/86(+), CD6(+), WC1(+), CD26(+) and expressed the myeloid markers CD205, CD172a and CD11a. In keeping with pDC, LIN(-) cells express TLR7 mRNA transcripts; however, in a resting state do not express TLR8 or TLR9. Functionally, LIN(-) cells, but not PBMC, monocytes and monocyte derived DC produce large amounts of IFNα/β in response to different CpG oligonucleotides. Taken together, we present data suggesting that an enriched circulating population of bovine LIN(-) cells are uniquely capable of producing IFNα/β in response to CpG oligonucleotides and thus this population likely contain the functional equivalent of bovine pDC., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Objective: The purpose of this study was to investigate changes of the antigen-presenting function of tumor infiltrating dendritic cells (TIDCs) in human endometrioid adenocarcinoma., Study Design: The TIDCs from 45 cases of endometrioid adenocarcinoma were compared with 20 cases of normal human endometrial tissue, using transmission electron microscopic examination, and the expression of CD80, CD86, and CD40 was analyzed by flow cytometry., Results: In comparison with the control group, the ultrastructure of TIDCs in human endometrioid adenocarcinoma showed the following differences: numerous TIDCs were small in volume and round in shape but some were oval and multi-angular. The cytoplasmic processes were obviously decreased in number and stubbed. Round primary lysosomes with high electron-dense granules, and secondary lysosomes with high or low electron-dense granules were seen frequently in the cytoplasm. TIDCs contained much rough endoplasmic reticulum (RER). Vacuoles with flocculent electron-dense granules were rare. High electron-dense contents in the granules were near one side and the other side was bright. The nucleus became markedly small in volume, nephroid or hoofed in shape. The nucleus had little euchromatin and lots of heterochromatin under the nuclear membrane. The levels of expression of CD80, CD86 and CD40 on TIDCs were low or even nonexistent. The expression levels of CD80, CD86 and CD40 on DCs in human normal endometrium were significantly higher than those on TIDCs in endometrioid adenocarcinoma., Conclusion: It is suggested that morphological differences and low expression of co-stimulatory molecules on TIDCs in endometrioid adenocarcinoma reflected the functional changes of the TIDCs in uptake, processing and presenting antigen, which may lead to the occurrence of tumor immune escape., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Dendritic cells were discovered and recognized as antigen-presenting cells in 1973. Since then, large volume of information has accumulated showing role of dendritic cells as a key element connecting the innate and adaptive immunity. Nowadays, dendritic cells are considered to be professional immune sensors capable of recognizing both antigen amounts and antigen persistence via complex mechanisms that involve decoding and integration of various signals received in a receptor-dependant manner. Tissue microenvironment plays an important role in the modulation of effector functions of dendritic cells leading either to activation or to suppression of immune reactions. Dendritic cells maintain the homeostasis and are involved in a number of diseases including infection diseases and cancer. The presence of dendritic cells in arteries has been reported in 1995. And since then the importance of dendritic cells in atherogenesis is intensively studied. This review briefly described current knowledge on dendritic cells and their role in atherogenesis.

Human mature dendritic cells (DCs) can efficiently stimulate natural killer (NK)-cell responses without being targeted by their cytotoxicity. To understand this important regulatory crosstalk, we characterized the development of the immunologic synapse between mature DCs and resting NK cells. Conjugates between these 2 innate leukocyte populations formed rapidly, persisted for prolonged time periods and matured with DC-derived f-actin polymerization at the synapse. Polarization of IL-12 and IL-12R to the synapse coincided with f-actin polymerization, while other activating and inhibitory molecules were enriched at the interface between DCs and NK cells earlier. Functional assays revealed that inhibition of f-actin polymerization in mature synapses led to an increase of IFN-γ secretion and cytotoxicity by NK cells. This elevated NK-cell reactivity resulted from decreased inhibitory signaling in the absence of MHC class I polarization at the interface, which was observed on inhibition of f-actin polymerization in DCs. Thus, inhibitory signaling is stabilized by f-actin at the synapse between mature DCs and resting NK cells.

The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.

HIV-1 cell-to-cell transmission confers a strong advantage as it increases efficiency of transfer up to 100-fold compared with a cell-free route. Mechanisms of HIV-1 cell-to-cell transmission are still unclear and can in part be explained by the presence of actin-containing cellular protrusions. Such protrusions have been shown to facilitate cell-to-cell viral dissemination. Using fluorescence microscopy, electron tomography, and ion abrasion scanning electron microscopy we show that HIV-1 induces membrane extensions in immature dendritic cells through activation of Cdc42. We demonstrate that these extensions are induced after engagement of DC-SIGN by HIV-1(env) via a cascade that involves Src kinases, Cdc42, Pak1, and Wasp. Silencing of Cdc42 or treatment with a specific Cdc42 inhibitor, Secramine A, dramatically reduced the number of membrane protrusions visualized on the cell surface and decreased HIV-1 transfer via infectious synapses. Ion abrasion scanning electron microscopy of cell-cell contact regions showed that cellular extensions from immature dendritic cells that have the appearance of thin filopodia in thin section images are indeed extended membranous sheets with a narrow cross section. Our results demonstrate that HIV-1 binding on immature dendritic cells enhances the formation of membrane extensions that facilitate HIV-1 transfer to CD4(+) T lymphocytes.

To investigate and analyze induction of phenotypic and functional maturation of murine DCs by GLP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DCs, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acid phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. It was found that GLP induced phenotypic maturation, as evidenced by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside the DCs, which occurs when phagocytosis of DCs decreased, and antigen presentation increased with maturation. Finally, GLP increased the production of IL-12. These data reveals that GLP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings that require a boosting of the immune response. Therefore concluded that GLP can exert positive induction to murine DCs at the used concentration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

One very striking feature of T-cell recognition is the formation of an immunological synapse between a T cell and a cell that it is recognizing. Formation of this complex structure correlates with cytotoxicity in the case of killer (largely CD8(+)) T-cell activity, or robust cytokine release and proliferation in the case of the much longer lived synapses formed by helper (CD4(+)) T cells. Here we have used electron microscopy and 3D tomography to characterize the synapses of antigen-specific CD4(+) T cells recognizing B cells and dendritic cells at different time points. We show that there are at least four distinct stages in synapse formation, proceeding over several hours, including an initial stage involving invasive T-cell pseudopodia that penetrate deeply into the antigen-presenting cell, almost to the nuclear envelope. This must involve considerable force and may serve to widen the search for potential ligands on the surface of the cell being recognized. We also show that centrioles and the Golgi complex are always located immediately beneath the synapse and that centrioles are significantly shifted toward the late contact zone with either B lymphocytes or bone marrow-derived dendritic cells such as antigen-presenting cells, and that there are dynamic, stage-dependent changes in the organization of microtubules beneath the synapse. These data reinforce and extend previous data on cytotoxic T cells that one of the principal functions of the immunological synapse is to facilitate cytokine secretion into the synaptic cleft, as well as provide important insights into the overall dynamics of this phenomenon.

SRS19-6MuLV is a member of the MuLV family originally isolated from the Tianjin-Shanghai-Zunyi complex of murine leukemia. A notable characteristic of this virus is that it induces tumors of multiple hematopoietic lineages, including myeloid, erythroid, T-lymphoid and B-lymphoid. In a previous study, a sequence with high homology to SRS19-6MuLV in a murine dendritic cell sarcoma (DCS) was identified through cDNA expression screening with mAb 983D4. To investigate the relationship between SRS19-6MuLV and DCS, the existence of a specific SRS19-6MuLV DNA fragment in DCS cells, 15 murine tumor cells, 2 murine tumor tissues, 12 normal murine cells/tissues, 11 human tumor cell lines and SRSV/3T3 (NIH/3T3 cells infected with SRS cell supernatant) was detected by PCR. The specific fragment of SRS19-6MuLV was detected in DCS, mouse fore-gastric cancer cells, LⅡ tumor tissue from which DCS is derived and SRSV/3T3. In addition, the integration sites of SRS19-6MuLV in the positive cells were examined by inverse PCR. Thus, 7 integration sites for SRS19-6MuLV were detected in DCS and 3 in SRSV/3T3. Analysis of sequences by BLAST revealed that some of the integration sites were associated with common fragile sites and some Ras-regulating miRNAs. Our results indicate that SRS19-6MuLV not only induced four types of leukemia, but also induced DCS. This virus does not infect human cells. Multiple integration of SRS19-6MuLV into chromosomes around fragile sites accounts for its carcinogenic effects.

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

In the steady state, hepatic antigen (Ag)-presenting cells (APC) generally dampen systemic inflammatory responses to gut-derived Ags. Our studies focus on the role of specific liver APC populations, both non-parenchymal cells (dendritic cells [DC], Kupffer cells, and hepatic stellate cells [HSC]) and parenchymal cells, in the molecular regulation of tissue damage (ischemia and reperfusion [I/R] injury) and immunity following liver transplantation. We focus on factors that either promote or overwhelm the natural tendency of the liver to suppress inflammatory/immune responses. We are also examining molecular mechanisms that regulate liver DC maturation and function and that determine their role in the control of allogeneic T-cell function and the fate of the transplanted liver. Our studies are also aimed at elucidating mechanisms by which HSC regulate DC and T-cell function. These investigations may provide new targets for therapeutic intervention in liver inflammation.

Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317(+) dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317(+) dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.

Kindlin-3 is a key lymphocyte function-associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3-null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3-null T lymphocytes failed to trigger the robust LFA-1-mediated T-cell spreading on ICAM-1 and ICAM-1-expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3-null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, β I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1-driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3-null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.

In this work, the capability of primary, monocyte-derived dendritic cells (DCs) to uptake iron oxide magnetic nanoparticles (MNPs) is assessed and a strategy to induce selective cell death in these MNP-loaded DCs using external alternating magnetic fields (AMFs) is reported. No significant decrease in the cell viability of MNP-loaded DCs, compared to the control samples, was observed after five days of culture. The number of MNPs incorporated into the cytoplasm was measured by magnetometry, which confirmed that 1-5 pg of the particles were uploaded per cell. The intracellular distribution of these MNPs, assessed by transmission electron microscopy, was found to be primarily inside the endosomic structures. These cells were then subjected to an AMF for 30 min and the viability of the blank DCs (i.e. without MNPs), which were used as control samples, remained essentially unaffected. However, a remarkable decrease of viability from approximately 90% to 2-5% of DCs previously loaded with MNPs was observed after the same 30 min exposure to an AMF. The same results were obtained using MNPs having either positive (NH(2)(+)) or negative (COOH(-)) surface functional groups. In spite of the massive cell death induced by application of AMF to MNP-loaded DCs, the number of incorporated magnetic particles did not raise the temperature of the cell culture. Clear morphological changes at the cell structure after magnetic field application were observed using scanning electron microscopy. Therefore, local damage produced by the MNPs could be the main mechanism for the selective cell death of MNP-loaded DCs under an AMF. Based on the ability of these cells to evade the reticuloendothelial system, these complexes combined with an AMF should be considered as a potentially powerful tool for tumour therapy.

Biodegradable poly-(D,L-lactide-co-glycolide) microspheres (PLGA-MS) are approved as a drug delivery system in humans and represent a promising antigen delivery device for immunotherapy against cancer. Immune responses following PLGA-MS vaccination require cross-presentation of encapsulated antigen by professional antigen presenting cells (APCs). While the potential of PLGA-MS as vaccine formulations is well established, the intracellular pathway of cross-presentation following phagocytosis of PLGA-MS is still under debate. A part of the controversy stems from the difficulty in unambiguously identifying PLGA-MS within cells. Here we show a novel strategy for the efficient encapsulation of inorganic nanocrystals (NCs) into PLGA-MS as a tool to study their intracellular localization. We microencapsulated NCs as an electron dense marker to study the intracellular localization of PLGA-MS by transmission electron microscopy (TEM) and as fluorescent labels for confocal laser scanning microscopy. Using this method, we found PLGA-MS to be rapidly taken up by dendritic cells and macrophages. Co-localization with the lysosomal marker LAMP1 showed a lysosomal storage of PLGA-MS for over two days after uptake, long after the initiation of cross-presentation had occurred. Our data argue against an escape of PLGA-MS from the endosome as has previously been suggested as a mechanism to facilitate cross-presentation of PLGA-MS encapsulated antigen., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The role of dendritic cells in vitiligo is still unclear. Few studies have provided contradictory results about their quantitative variation and no data exist concerning their immunophenotypical distribution in diseased skin. The purpose of our study was to analyze the presence, the distribution, the immunophenotypical markers and the effects of nb-UVB therapy on dendritic cells in non-lesional, perilesional, and lesional vitiligo skin. Punch-biopsies of 6 mm were taken from lesional, perilesional, and non-lesional skin of 12 patients affected by non-segmental vitiligo, treated with nb-UVB. An immunohistochemical and an ultrastructural analysis were performed. Immunohistochemical and ultrastructural analysis showed both quantitative and qualitative modifications of Langerhans cells. Nb-UVB therapy, one of the most effective treatments for the disease, was able to reduce the Langerhans cells number and to redistribute main dendritic subsets. This study underlines the importance of dendritic cells, Langerhans cells in particular, in non-segmental vitiligo, in its pathogenesis and in its better therapeutical approach.

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall., (Copyright © 2010 Elsevier B.V. All rights reserved.)

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.

Cross-presentation is the key process in stimulation of cytotoxic T lymphocyte (CTL) immune response in eliminating many infectious diseases and tumors. Previous studies have shown that surface modification of poly(D,L-lactic-co-glycolic acid) (PLGA) particles with polycations enhanced their adjuvant ability resulting in a strong antibody response to the encapsulated antigen. However, the in vitro cross-presentation by protamine-coated PLGA nanoparticles (NPs) has not been addressed yet. In this study, a model antigen ovalbumin (OVA) was encapsulated into PLGA nanoparticles, with (OVA-NPs/protamine) or without protamine coating (OVA-NPs). These nanoparticles were then used to stimulate murine bone marrow-derived dendritic cells (BMDCs). Flow cytometry analysis revealed an increase in endocytosis of protamine-coated PLGA nanoparticles by BMDCs at 37°C. Compared with OVA-NPs-treated BMDCs, stimulation with OVA-NPs/protamine led to significantly upregulation of CD80, CD86, and CD83, increased secretion of IL-12p70, and decreased production of IL-4 by BMDCs. Furthermore, OVA-NPs/protamine-treated BMDCs also showed an enhanced cross-presentation to B3Z T cell hybridoma in vitro. Transmission electron microscopy (TEM) study showed that protamine-coated PLGA nanoparticles escaped from lysosomes through the interaction with lysosomal membrane. These results demonstrated that protamine-coated PLGA nanoparticles could enhance the cross-presentation of encapsulated exogenous antigen by facilitating antigen uptake and lysosomal escape, suggesting the feasibility to be a potent adjuvant for cellular vaccines., (Copyright © 2010 Wiley Periodicals, Inc.)

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.

In the last decade, it has been accepted the formation of tertiary lymphoid organs in the renal parenchyma during inflammatory conditions. These organized cellular aggregates contain B- and T-lymphocytes, dendritic cells, surrounded by neo-lymphatic vessels. They have been described in renal allografts, acute and chronic interstitial nephritis, IgA and membranous nephropathies. The functional characteristics of these lymphoid nodules remained still under consideration. After investigating the renal biopsies of 268 patients with primary and secondary nephropathies, we have selected 20 cases showing lymphoid-like cellular aggregates located just beneath the renal capsule and having close contacts with this kidney envelope. All of these cases also showed an associated medium sized lymphatic vessel. The ultrastructure of these nodules proved to contain more or less the same cellular composition: lymphocytes, dendritic cells, seldom plasma cells and macrophages. We consider these particular subcapsular lymphoid-like nodules to be tertiary lymphatic structures in close association with the perirenal lymphatics, and the first to develop in any type of inflammatory and autoimmune renal condition.

Laser correlation spectroscopy, atomic force microscopy, and immunoaffinity chromatography were used to characterize exosomes produced by different human cells. The exosomes secreted into a culture medium by normal fibroblasts, dendritic cells, lymphocytes, as well as malignant cells obtained from tumors of various tissue origins. The similar investigations were made for exosomes detectable in plasma and cerebrospinal fluid. The dynamic light scattering technique has demonstrated that the exosomes from different sources are homogenous and similar in size of the order of 20 and 90 nm. The exceptional homogeneity of exosomes was confirmed by atomic force microscopy. The immunoaffinity method has shown that all the exosomes under study carry antigenic determinants recognizable by antibodies to the major histocompatibility complex of type 1 (HLA-ABC). A method is proposed for evidence-based detection of exosomes in various biological fluids. For this, dynamic light scattering detects 20- and 90-nm particles and whether they can be removed by immunoaffinity chromatography with HLA-ABC antibodies is checked.

Confocal Raman micro-spectroscopy (CRMS) was used to measure spectral images of immunological synapse formation between dendritic and T cells without using molecular labels or other invasive procedures. The purpose-built inverted CRMS instrument integrated an environmental enclosure and a near-infrared laser to allow measurements on live cells maintained under physiological conditions. The integration of the wide-field fluorescence also enabled viability assays and direct comparison between Raman spectral images and gold-standard immuno-fluorescence images for specific molecules. Raman spectral images of nucleus and proteins were built by fuzzy c-mean clustering method. The Raman images were found to be in good correspondence with the immuno-fluorescence images of DNA and actin. These results indicate that actin is a main contributor to the Raman spectrum of the cytoplasm of dendritic and T cells. While for control cells the Raman spectral images of proteins indicated a more homogeneous distribution of proteins in the cytoplasm of dendritic cells, they indicated a higher accumulation of proteins at the immunological synapses when dendritic cells were pre-treated with laminin. These conclusions were also supported by confocal immuno-fluorescence imaging after cell fixation and labelling. This study demonstrates the potential of CRMS for label-free non-invasive imaging of junctions between live cells. Therefore, this technique may become a useful tool for studying cellular processes in live cells and where non-invasive molecular specific imaging is desirable, such as cell-cell interactions.

Nanoparticles (NPs) have been reported to penetrate into human skin through lesional skin or follicular structures. Therefore, their ability to interact with dendritic cell (DC) was investigated using DCs generated from monocytes (mono-DCs). Hybrid titanium dioxide/para-amino benzoic acid (TiO(2)/PABA) NPs did not induce any cell toxicity. NPs were internalised into DCs through macropinocytosis and not by a receptor-mediated mechanism. Confocal microscopy showed that NPs were not detected in the nucleus. These data are confirmed by electronic microscopy which demonstrated that hybrid NPs were rapidly in contact with cellular membrane and localised into cytoplasmic vesicles without colocalisation with clathrin-coated vesicles. Hybrid NPs did not induce CD86 or HLA-DR overexpression or cytokine secretion (IL-8 and TNF-α) indicating no DC activation. Internalisation of hybrid NPs did not modify DC response towards sensitisers such as nickel and thimerosal or LPS used as positive controls. Moreover, hybrid NPs did not induce any oxidative stress implicated in DC activation process. After mono-DC irradiation by ultraviolet A (UVA), hybrid NP-treated cells did not produce UVA-induced reactive oxygen species (ROS) and exhibited a better cell viability compared with UVA-irradiated control cells, suggesting a protecting effect of hybrid TiO(2)/PABA NPs against UVA-induced ROS., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306-318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell-cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC-DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.

Background: The possible therapeutic benefits of using steroids to enhance muscle strength and slow disease progression in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) has been examined previously. In this investigation, it was hypothesised that steroid therapy is associated with morphological changes in the dystrophic muscle., Objectives and Methods: To test this hypothesis, two muscle biopsies were obtained (one biopsy before treatment, and the second 6 months following prednisone therapy) from 24 patients with dystrophies (18 DMD, 6 BMD). The participants were categorised into: control (6 specimens, normal muscle), untreated and treated groups. The muscle was evaluated for ultrastructural changes using transmission electron microscopy (TEM)., Results: In the untreated group, the muscle fibres were degenerated and of variable sizes. The myofibrils were thin with either complete loss of bands and/or abnormal banding patterns. The Z-lines were irregularly spaced and loosely registered. The mitochondria of the myofibrils were small, few, spherical and irregularly distributed. Numerous dendritic cells (DCs) with euchromatic nuclei, and multiple and long dendrites, were seen among the myofibrils. The collagen fibres among the muscle fibres (endomysium) were numerous and large. The satellite cells had euchromatic nuclei with clumps of heterochromatin. In the treated group, the muscle fibres had a relatively uniform size with occasional fibres showing partial degeneration. The myofibrils had a relatively similar diameter comparable to that of normal muscle .The degenerated areas were small in size with occasional foci showing loss of banding pattern, and abnormal short bands with thick and hazy Z-lines. The mitochondria of the myofibrils were numerous, spherical, small in size and regularly arranged between the myofibrils. Few DCs, with heterochromatic nuclei, and few and short dendrites appeared between the myofibrils. The collagen fibres between the muscle fibres (endomysium) were numerous and large. As compared with the treated group, there was a statistically significant increase (p<0.05) in the numbers of DCs (0.7+/-0.2 vs 1.6+/-0.3) and fibroblasts (1.9+/-0.2 vs 2.9 +/-0.3) in the untreated group. Alternatively, there was a statistically significant decrease (p<0.05) in the numbers of satellite cells (1.2+/-0.2 vs 0.6+/-0.1)., Conclusion: The ability of steroids to induce ultrastructural features of improvement supports the notion that they have beneficial therapeutic role. The clinical ramifications of these observations mandate further studies.

Dendritic cells (DC) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes and initiate primary immune responses. Angiotensin II (AII) is involved in key events of the inflammatory response. Because our previous work implicated an effect of AII on differentiation and function of murine and human DC, we investigated the impact of AII type 1 receptor (AT(1)) deficiency on the phenotypical and functional properties of mouse DC in vitro and in vivo. Bone marrow (BM) cells isolated from mice lacking AII subtype 1a receptor (AT(1a)), AII subtype 1b receptor (AT(1b)), or both receptor isoforms and control littermates [wild type (WT)] were cultured for 7 days in the presence of recombinant mouse granulocyte/macrophage colony-stimulating factor to generate myeloid DC in vitro. Generation of CD11c(+) cells was less efficient in both AT(1a)- and AT(1b)-deficient BM cells than in WT BM cell cultures. Moreover, DC generated from AT(1)-deficient progenitors showed lower levels of expression of major histocompatibility complex II (MHC-II) and CD11c (p < 0.01) and a marked reduction in their allostimulatory activity (p < 0.01 or 0.001). Although AT(1)-deficient DC released comparable levels of interleukin (IL)-10 and IL-12p70 to WT DC, they produced significantly lower levels of tumor necrosis factor alpha (TNF-alpha) (p < 0.05). Remarkably, CD11c(+) cells isolated from the spleen of AT(1) knockout mice challenged with lipopolysaccharide in vivo up-regulated MHC-II, CD40, and CD80 as did WT, but released significantly lower levels of TNF-alpha (p < 0.01). These data provide clear evidence that AT(1) controls differentiation and functionality of DC and thus may have a crucial impact on inflammatory processes where local angiotensinergic systems are known to be activated.

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

To investigate whether tumor cell lysate-pulsed (TP) dendritic cells (DCs) induce cytotoxic T lymphocyte (CTL) activity against colon cancer in vitro and in vivo. Hematopoietic progenitor cells were magnetically isolated from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFalpha to induce their maturation. They were analyzed by morphological observation and phenotype analysis. DCs were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. CTL activity and interferon gamma (IFNgamma) secretion was evaluated ex vivo. In order to determine whether or not vaccination with CT26 TP DCs induce the therapeutic potential in the established colon tumor model, CT26 colon tumor cells were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice. Tumor-bearing mice were injected with vaccination with CT26 TP DCs on days 3 and 10. Tumor growth was assessed every 2-3 days. Finally, CTL activity and IFNgamma secretion were evaluated in immunized mice. Hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed high levels of MHC II, CD11b, CD80, and CD86 antigen, and were negative for CD8alpha. However, immature DCs cultured with cytokines for 5 days did not have typical DCs phenotypic markers. Ex vivo primed T cells with CT26 TP DCs were able to induce effective CTL activity against CT26 tumor cells, but not B16 tumor cells (E:T = 100:1, 60.36 +/- 7.11% specific lysis in CT26 group vs. 17.36 +/- 4.10% specific lysis in B16 group), and produced higher levels of IFNgamma when stimulated with CT26 tumor cells but not when stimulated with B16 tumor cells (1210.33 +/- 72.15 pg/ml in CT26 group vs. 182.25 +/- 25.51 pg/ml in B16 group, P < 0.01). Vaccination with CT26 TP DCs could induce anti-tumor immunity against CT26 colon tumor in murine therapeutic models (tumor volume on day 19: CT26 TP DCs 342 +/- 55 mm(3) vs. the other control groups, P < 0.05). In addition, all splenic CD3(+) T cells obtained from mice vaccinated with CT26 TP DCs produced high levels of IFNgamma and shown specific cytotoxic activity against CT26 tumor cells, but no cytotoxic activity when stimulated with B16 tumor cells. Tumor cell lysate-pulsed DCs can induce tumor-specific CTL activity against colon cancer in vitro and in vivo.

The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells with the unique ability of primary immune response initiation. DCs originate from bone marrow progenitors, which circulate in the peripheral blood and subsequently penetrate peripheral tissues, where they give rise to immature DCs. In peripheral tissues, DCs continuously monitor the microenvironment and, when the cells encounter 'danger' signals, DCs undergo differentiation and maturation. Maturing DCs usually migrate to lymphatic tissues, where they form contacts with T cells to initiate a primary immune response. DCs were identified in arteries in 1995 and since then, further knowledge has been gained about the peculiarities of vascular-associated DCs and their role in atherosclerosis. Immune reactions toward modified lipoproteins and other factors ignited by resident vascular DCs as well as by newly arrived DCs, which originate from blood monocytes, are believed to destabilize arterial homeostasis from very earlier stages of atherogenesis. There is a remarkable heterogeneity of DCs in atherosclerotic lesions. Some DCs mature and become capable of forming clusters with T cells directly within the arterial wall. The predictive value of the numbers of circulating DC precursors in coronary artery disease and in atherosclerosis has been assessed, and it has been shown that DCs have a role in plaque destabilization. Over recent decades, DCs have proven to be a valuable instrument in immunotherapy approaches against cancer and various autoimmune diseases, and this explains the demand that the accumulated knowledge be applied to the field of atherosclerosis immunotherapy.

Human monocyte-derived dendritic cells (mo-DCs) express highly sialylated structures, with recognized but poorly understood function in maturation, immunogenicity and endocytosis capacity. We have previously shown that mo-DCs surface sialylation is changeable upon different stimuli, which led us to hypothesise the existence of cell surface (non-intracellular) sialyltransferases, rapidly restoring or altering mo-DC surface sialylation, thus modulating specific functions. Here, we demonstrate that, in the presence of exogenous CMP-Neu5Ac, mo-DCs incorporate considerable amounts of sialic acids into cell surface, predominantly when mo-DCs were previously desialylated or matured. This is a genuine sialyltransferase activity, confirmed by specific inhibition assays, which is not influenced by secreted enzymes. Functionally, the ecto-sialyltransferase activity causes a significant down-regulation of mo-DCs endocytic capacity, without affecting the maturation state. These findings suggest that ecto-sialyltransferases participate in a dynamic control of mo-DC sialylation, with functional repercussions. This activity is possibly related with specific physiological and pathological conditions, as inflammation and infection, contributing to protection and homeostasis regulation., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Human herpesvirus 6B (HHV-6B) is the causative agent of the common childhood febrile illness, exanthema subitum. The virus is predominantly regarded as a T-cell tropic virus, although in reality it has the ability to infect a wide variety of cell types including monocytes, macrophages and dendritic cells (DC). Although DC are important immune regulators, the modulating effects of HHV-6B on DC are controversial. Here, we examine the phenotypic and functional consequences of HHV-6B infection of DC. The addition of HHV-6B to immature DC led to expression of the nuclear viral p41 protein and cell surface expression of the viral glycoprotein gp60/110 consistent with HHV-6B infection. Nevertheless, HHV-6B did not induce noticeable cytopathogenic effects or cell death in infected DC. Importantly, HHV-6B infection induced a partial phenotypic maturation of immature DC as demonstrated by a substantial increase in the expression of HLA-DR, CD86 and CD40, whereas only a minor increase in CD80 and CD83 was observed. This phenotypic maturation was, however, not followed by functional maturation, because HHV-6B infection did not induce IL-10 and IL-12p70 production in immature DC. However, infected DC were still able to react to bacteria-derived stimuli such as lipopolysaccaharide by an even more pronounced production of IL-10 and IL-12p70 when compared to that of uninfected DC.

Background: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes., Methodology/principal Findings: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection., Conclusions/significance: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host.

Unlabelled: Nanobodies are single-domain antigen-binding fragments derived from heavy-chain antibodies that are devoid of light chains and occur naturally in Camelidae. We have shown before that their small size and high affinity and specificity for their target antigen make Nanobodies ideal probes for in vivo tumor imaging. In the present study, we have evaluated the use of Nanobodies as a generic method for imaging the in vivo biodistribution of specific immune cell types, using myeloid cells as an example., Methods: The cellular specificity of Nanobodies raised against murine bone marrow-derived dendritic cells was verified using flow cytometry on a range of myeloid and nonmyeloid cell types. The Nanobodies were then labeled with (99m)Tc and their biodistribution was analyzed using SPECT. The biodistribution was also assessed by measuring radioactivity in various organs and tissues. To verify whether the observed biodistribution was due to specific targeting through the antigen-binding loops, rather than retention in organs because of effects of the framework regions, we genetically grafted the antigen-binding loops of the Nanobodies onto the framework region of a Nanobody scaffold that by itself showed low background retention in the periphery. The cellular specificity and biodistribution of these grafted Nanobodies were determined as before., Results: Nb-DC2.1, which recognizes a wide range of myeloid cells, targets most strongly to the liver, spleen, and lungs. Nb-DC1.8, which recognizes immature bone marrow-derived dendritic cells in vitro, gives a much smaller signal in the liver and spleen than does Nb-DC2.1 but mainly targets to the lungs and gives a pronounced signal in the skin. Grafting of the antigen-binding loops of Nb-DC1.8 or Nb-DC2.1 to the scaffold of Nb-BCII10 alters the observed biodistribution of the Nanobodies to resemble that of the Nanobody from which the antigen-binding loops have been derived., Conclusion: The observed in vivo biodistribution of the Nanobodies reflects the main in vivo locations of the cells recognized by the Nanobodies and is determined by the antigen-binding loops of the Nanobodies. Thus, Nanobodies represent elegant targeting probes for imaging the in vivo biodistribution of specific immune cell types.

Podosomes are spot-like actin-rich structures formed at the ventral surface of monocytic and haematopoietic cells. Podosomes degrade extracellular matrix and are proposed to be involved in cell migration. A key question is whether podosomes form protrusions similar to the invadopodia of cancer cells. We characterised podosomes of immature dendritic cells using electron microscopy combined with both conventional and novel high-resolution structured illumination light microscopy. Dendritic cell podosomes are composed of actin foci surrounded by a specialised ring region that is rich in material containing paxillin. We found that podosomes were preferential sites for protrusion into polycarbonate filters impregnated with crosslinked gelatin, degrading up to 2 micrometers of matrix in 24 hours. Podosome-associated uptake of colloidal gold-labelled gelatin matrix appeared to occur via large phagosome-like structures or narrow tubular invaginations. The motor protein myosin-II was excluded from ring or core regions but was concentrated around them and the myosin-II inhibitor Blebbistatin reduced the length of podosome protrusions. Finally, we found that degradation, protrusion and endocytosis in this system are dependent on the matrix metalloproteinase MMP-14. We propose that podosomes mediate migration of dendritic cells through tissues by means of myosin-II-dependent protrusion coupled to MMP-14-dependent degradation and endocytosis.

Background: Dendritic cells (DCs) are applied worldwide in several clinical studies of immune therapy of malignancies, autoimmune diseases, and transplantations. Most legislative bodies are demanding high standards for cultivation and transduction of cells. Closed-cell cultivating systems like cell culture bags would simplify and greatly improve the ability to reach these cultivation standards. We investigated if a new polyolefin cell culture bag enables maturation and adenoviral modification of human DCs in a closed system and compare the results with standard polystyrene flasks., Study Design and Methods: Mononuclear cells were isolated from HLA-A*0201-positive blood donors by leukapheresis. A commercially available separation system (CliniMACS, Miltenyi Biotec) was used to isolate monocytes by positive selection using CD14-specific immunomagnetic beads. The essentially homogenous starting cell population was cultivated in the presence of granulocyte-macrophage-colony-stimulating factor and interleukin-4 in a closed-bag system in parallel to the standard flask cultivation system. Genetic modification was performed on Day 4. After induction of maturation on Day 5, mature DCs could be harvested and cryopreserved on Day 7. During the cultivation period comparative quality control was performed using flow cytometry, gene expression profiling, and functional assays., Results: Both flasks and bags generated mature genetically modified DCs in similar yields. Surface membrane markers, expression profiles, and functional testing results were comparable. The use of a closed-bag system facilitated clinical applicability of genetically modified DCs., Conclusions: The polyolefin bag-based culture system yields DCs qualitatively and quantitatively comparable to the standard flask preparation. All steps including cryopreservation can be performed in a closed system facilitating standardized, safe, and reproducible preparation of therapeutic cells.

Exosomes are nanovesicles originating from late endosomal compartments and secreted by most living cells in ex vivo cell culture conditions. The interest in exosomes was rekindled when B-cell and dendritic cell-derived exosomes were shown to mediate MHC-dependent immune responses. Despite limited understanding of exosome biogenesis and physiological relevance, accumulating evidence points to their bioactivity culminating in clinical applications in cancer. This review focuses on the preclinical studies exploiting the immunogenicity of dendritic cell-derived exosomes (Dex) and will elaborate on the past and future vaccination trials conducted using Dex strategy in melanoma and non-small cell lung cancer patients.

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the "fungipod", formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7+/-5.6 microm long and 1.8+/-0.67 microm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225+/-55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 microm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion.