Human Dendritic Cells Research Articles

Mechanism of human peripheral blood-derived dendritic cells in the regulation of autophagy in carcinoma cells.

Dendritic cells (DC) are involved in immune recognition, response and immunomodulation mechanisms related to the onset of cancer. To explore DCs mechanism in the inhibition of autophagy in hepatoma cells. Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and induced into DCs, which were co-cultured with HepG2 cells by Transwell migration assay. HepG2 cell activity was determined using the CCK8 assay. LC3 autophagy index expression was measured with Western blot analysis, and the expression and secretion of cytokines, with qRT-PCR and ELISA. In the co-culture system, DCs were able to reduce HepG2 cells viability; IL-2, IL-12, IL-10 and IFN-γ expression in DCs was also significantly inhibited, although IL-2 and IFN-γ were still expressed 0.6 and 0.53 more than in the control group. DCs can regulate autophagy in hepatocellular carcinoma cells. The mechanism may be related to the synthesis and release of cytokines such as IL-2, IL-12 and IFN-γ by DCs.

Extra-viral DNA in adeno-associated viral vector preparations induces TLR9-dependent innate immune responses in human plasmacytoid dendritic cells

Adeno-associated viral (AAV) vector suspensions produced in either human derived HEK cells or in Spodoptera frugiperda (Sf9) insect cells differ in terms of residual host cell components as well as species-specific post-translational modifications displayed on the AAV capsid proteins. Here we analysed the impact of these differences on the immunogenic properties of the vector. We stimulated human plasmacytoid dendritic cells with various lots of HEK cell-produced and Sf9 cell-produced AAV-CMV-eGFP vectors derived from different manufacturers. We found that AAV8-CMV-eGFP as well as AAV2-CMV-eGFP vectors induced lot-specific but not production platform-specific or manufacturer-specific inflammatory cytokine responses. These could be reduced or abolished by blocking toll-like receptor 9 signalling or by enzymatically reducing DNA in the vector lots using DNase. Successful HEK cell transduction by DNase-treated AAV lots and DNA analyses demonstrated that DNase did not affect the integrity of the vector but degraded extra-viral DNA. We conclude that both HEK- and Sf9-cell derived AAV preparations can contain immunogenic extra-viral DNA components which can trigger lot-specific inflammatory immune responses. This suggests that improved strategies to remove extra-viral DNA impurities may be instrumental in reducing the immunogenic properties of AAV vector preparations.

Complement protein C3a enhances adaptive immune responses towards FVIII products.

The most serious complication in the treatment of hemophilia A (HA) is the development of factor (F)VIII inhibitors or antidrug antibodies (ADA) occurring in 25-35% of patients with severe HA. The immunological mechanisms underlying the development of ADA against FVIII products have not been completely understood yet. Immunological danger signals associated with events such as infection or surgery have been suggested to play a critical role. In previous studies, we demonstrated that plasma-derived (pd)FVIII but not recombinant (r)FVIII can activate human monocyte-derived dendritic cells (DC) in a danger signal-dependent manner, which subsequently mediate the proliferation of autologous CD4+ T cells. In this study, we investigated the ability of plasma components, naturally present in pdFVIII products, to mediate T-cell responses. In fact, we show that addition of plasma to rFVIII plus lipopolysaccharide (LPS)-stimulated DC induces proliferation of autologous CD4+ T cells. Interestingly, although DC pulsed with LPS plus plasma induce T-cell proliferation upon co-culture, the addition of FVIII significantly increases the number of proliferating as well as FVIII-specific CD4+ T cells. Total proliferating CD4+ T cells and FVIII-specific subsets were identified mainly as central memory T cells. Experiments using blocking antibodies and receptor antagonists revealed that the complement proteins C3a and, to a lesser extent, C5a are critically involved in these LPS-mediated T-cell responses. Collectively, our results indicate that complement proteins are potent drivers of T-cell responses to FVIII. Data presented provide a model how event-related substitution of FVIII in HA patients might contribute to inhibitor development.

High extracellular sodium chloride concentrations induce resistance to LPS signal in human dendritic cells.

Recent evidence showed that in response to elevated sodium dietary intakes, many body tissues retain Na+ ions for long periods of time and can reach concentrations up to 200mM. This could modulate the immune system and be responsible for several diseases. However, studies brought contrasted results and the effects of external sodium on human dendritic cell (DC) responses to danger signals remain largely unknown. Considering their central role in triggering T cell response, we tested how NaCl-enriched medium influences human DCs properties. We found that DCs submitted to high extracellular Na+ concentrations up to 200mM remain viable and maintain the expression of specific DC markers, however, their maturation, chemotaxis toward CCL19, production of pro-inflammatory cytokines and ROS in response to LPS were also partially inhibited. In line with these results, the T-cell allostimulatory capacity of DCs was also inhibited. Finally, our data indicate that high NaCl concentrations triggered the phosphorylation of SGK1 and ERK1/2 kinases. These results raised the possibility that the previously reported pro-inflammatory effects of high NaCl concentrations on T cells might be counterbalanced by a downregulation of DC activation.

Endo-lysosomal assembly variations among human leukocyte antigen class I (HLA class I) allotypes.

The extreme polymorphisms of human leukocyte antigen class I (HLA class I) proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes. The canonical endoplasmic reticulum (ER) HLA class I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA class I molecules. We hypothesized variable influences of HLA class I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA class I molecules are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B*08:01 or HLA-B*35:01 being among the most resistant or sensitive, respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in accumulation of HLA-B*35:01 in LAMP1+ compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA class I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA class I expression and increase the efficiency of cross-presentation.

Comparing the Biological Properties of Double-Stranded DNA Extracted from Human and Porcine Placenta and Salmon Sperm.

Double-stranded fragmented extracellular DNA is a participant, inducer, and indicator of various processes occurring in the organism. When investigating the properties of extracellular DNA, the question regarding the specificity of exposure to DNA from different sources has always been raised. The aim of this study was to perform comparative assessment of biological properties of double-stranded DNA obtained from the human placenta, porcine placenta and salmon sperm. The intensity of leukocyte-stimulating effect of different dsDNA was assessed in mice after cyclophosphamide-induced cytoreduction. The stimulatory effect of different dsDNA on maturation and functions of human dendritic cells and the intensity of cytokine production by human whole blood cells was analyzed ex vivo. The oxidation level of the dsDNA was also compared. Human placental DNA exhibited the strongest leukocyte-stimulating effect. DNA extracted from human and porcine placenta exhibited similar stimulatory action on maturation of dendritic cells, allostimulatory capacity, and ability of dendritic cells to induce generation of cytotoxic CD8+CD107a+ T cells in the mixed leukocyte reaction. DNA extracted from salmon sperm stimulated the maturation of dendritic cells, while having no effect on their allostimulatory capacity. DNA extracted from human and porcine placenta was shown to exhibit a stimulatory effect on cytokine secretion by human whole blood cells. The observed differences between the DNA preparations can be caused by the total methylation level and are not related to differences in oxidation level of DNA molecules. Human placental DNA exhibited the maximum combination of all biological effects.

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system.

Several vaccine platforms have been developed to fight pathogenic threats, with Virus-Like Particles (VLPs) representing a very promising alternative to traditional platforms. VLPs trigger strong and lasting humoral and cellular immune responses with fewer safety concerns and higher stability than other platforms. The use of extensively characterized carrier VLPs modified with heterologous antigens was proposed to circumvent the viral complexity of specific viruses that could lead to poor VLP assembly and yields. Although carrier VLPs have been successfully produced in a wide variety of cell-based systems, these are limited by low protein yields and protracted clone selection and optimization workflows that limit VLP screening approaches. In response, we have demonstrated the cell-free protein synthesis (CFPS) of several variants of the hepatitis B core (HBc) carrier VLP using a high-yielding tobacco BY-2 lysate (BYL). High VLP yields in the BYL system allowed in-depth characterization of HBc variants. Insertion of heterologous sequences at the spike region of the HBc monomer proved more structurally demanding than at the N-terminus but removal of the C-terminal domain allowed higher particle flexibility and insert acceptance, albeit at the expense of thermal and chemical stability. We also proved the possibility to scale the CFPS reaction up to 1L in batch mode to produce 0.45 grams of the native HBc VLP within a 48-hour reaction window. A maximum yield of 820 µg/ml of assembled VLP particles was observed at the 100µl scale and most remarkably the CFPS reaction was successfully scaled from 50µl to 1L without any reduction in protein yield across this 20,000-fold difference in reaction volumes. We subsequently proved the immunogenicity of BYL-derived VLPs, as flow cytometry and microscopy clearly showed prompt recognition and endocytosis of fluorescently labelled VLPs by human dendritic cells. Triggering of inflammatory cytokine production in human peripheral blood mononuclear cells was also quantitated using a multiplex assay. This research establishes BYL as a tool for rapid production and microscale screening of VLP variants with subsequent manufacturing possibilities across scales, thus accelerating discovery and implementation of new vaccine candidates using carrier VLPs.

Dendritic cell phenotype and function in a 3D co-culture model of patient-derived metastatic colorectal cancer organoids.

Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.

Single-cell RNA-seq analysis reveals dual sensing of HIV-1 in blood Axl+ dendritic cells

Sensing of incoming viruses is a pivotal task of dendritic cells (DCs). Human primary blood DCs encompass various subsets that are diverse in their susceptibility and response to HIV-1. The recent identification of the blood Axl+DC subset, endowed with unique capacities to bind, replicate, and transmit HIV-1 prompted us to evaluate its anti-viral response. We demonstrate that HIV-1 induced two main broad and intense transcriptional programs in different Axl+DCs potentially induced by different sensors; an NF-κB-mediated program that led to DC maturation and efficient CD4+ Tcell activation, and a program mediated by STAT1/2 that activated type I IFN and ISG responses. These responses were absent from cDC2 exposed to HIV-1 except when viral replication was allowed. Finally, Axl+DCs actively replicating HIV-1 identified by quantification of viral transcripts exhibited a mixed NF-κB/ISG innate response. Our results suggest that the route of HIV-1 entry may dictate different innate sensing pathways by DCs.

Glutamine synthetase expression rescues human dendritic cell survival in a glutamine-deprived environment.

Glutamine deficiency is a well-known feature of the tumor environment. Here we analyzed the impact of glutamine deprivation on human myeloid cell survival and function. Different types of myeloid cells were cultured in the absence or presence of glutamine and/or with L-methionine-S-sulfoximine (MSO), an irreversible glutamine synthetase (GS) inhibitor. GS expression was analyzed on mRNA and protein level. GS activity and the conversion of glutamate to glutamine by myeloid cells was followed by 13C tracing analyses. The absence of extracellular glutamine only slightly affected postmitotic human monocyte to dendritic cell (DC) differentiation, function and survival. Similar results were obtained for monocyte-derived macrophages. In contrast, proliferation of the monocytic leukemia cell line THP-1 was significantly suppressed. While macrophages exhibited high constitutive GS expression, glutamine deprivation induced GS in DC and THP-1. Accordingly, proliferation of THP-1 was rescued by addition of the GS substrate glutamate and 13C tracing analyses revealed conversion of glutamate to glutamine. Supplementation with the GS inhibitor MSO reduced the survival of DC and macrophages and counteracted the proliferation rescue of THP-1 by glutamate. Our results show that GS supports myeloid cell survival in a glutamine poor environment. Notably, in addition to suppressing proliferation and survival of tumor cells, the blockade of GS also targets immune cells such as DCs and macrophages.

The Monocytic Cell Line THP-1 as a Validated and Robust Surrogate Model for Human Dendritic Cells.

We have implemented an improved, cost-effective, and highly reproducible protocol for a simple and rapid differentiation of the human leukemia monocytic cell line THP-1 into surrogates for immature dendritic cells (iDCs) or mature dendritic cells (mDCs). The successful differentiation of THP-1 cells into iDCs was determined by high numbers of cells expressing the DC activation markers CD54 (88%) and CD86 (61%), and the absence of the maturation marker CD83. The THP-1-derived mDCs are characterized by high numbers of cells expressing CD54 (99%), CD86 (73%), and the phagocytosis marker CD11b (49%) and, in contrast to THP-1-derived iDCs, CD83 (35%) and the migration marker CXCR4 (70%). Treatment of iDCs with sensitizers, such as NiSO4 and DNCB, led to high expression of CD54 (97%/98%; GMFI, 3.0/3.2-fold induction) and CD86 (64%/96%; GMFI, 4.3/3.2-fold induction) compared to undifferentiated sensitizer-treated THP-1 (CD54, 98%/98%; CD86, 55%/96%). Thus, our iDCs are highly suitable for toxicological studies identifying potential sensitizing or inflammatory compounds. Furthermore, the expression of CD11b, CD83, and CXCR4 on our iDC and mDC surrogates could allow studies investigating the molecular mechanisms of dendritic cell maturation, phagocytosis, migration, and their use as therapeutic targets in various disorders, such as sensitization, inflammation, and cancer.

Increased cross-presentation by dendritic cells and enhanced anti-tumour therapy using the Arp2/3 inhibitor CK666

BackgroundDendritic cell (DC) vaccines for cancer therapy offer the possibility to let the patient’s own immune system kill cancer cells. However, DC vaccines have shown less efficacy than expected due to failure to induce cancer cell killing and by activating T regulatory cells.MethodsWe tested if inhibition of signalling via WASp and Arp2/3 using the small molecule CK666 would enhance DC-mediated killing of tumour cells in vitro and in vivo.ResultsUsing CK666 during the ex vivo phase of antigen processing of ovalbumin (OVA), murine and human DCs showed decreased phagosomal acidification, indicating activation of the cross-presentation pathway. When compared to untreated DCs, DCs treated with CK666 during uptake and processing of OVA-induced increased proliferation of OVA-specific CD8+ OT-I T cells in vitro and in vivo. Using the aggressive B16-mOVA melanoma tumour model, we show that mice injected with CK666-treated DCs and OVA-specific CD8+ OT-I T cells showed higher rejection of B16 melanoma cells when compared to mice receiving non-treated DCs. This resulted in the prolonged survival of tumour-bearing mice receiving CK666-treated DCs. Moreover, combining CK666-treated DCs with the checkpoint inhibitor anti-PD1 further prolonged survival.ConclusionOur data suggest that the small molecule inhibitor CK666 is a good candidate to enhance DC cross-presentation for cancer therapy.

Gram-Positive Bacteria Cell Wall Peptidoglycan Polymers Activate Human Dendritic Cells to Produce IL-23 and IL-1β and Promote TH17 Cell Differentiation.

Gram-positive bacterial infections are a major cause of organ failure and mortality in sepsis. Cell wall peptidoglycan (PGN) is shed during bacterial replication, and Bacillus anthracis PGN promotes a sepsis-like pathology in baboons. Herein, we determined the ability of polymeric Bacillus anthracis PGN free from TLR ligands to shape human dendritic cell (DC) responses that are important for the initiation of T cell immunity. Monocyte-derived DCs from healthy donors were incubated with PGN polymers isolated from Bacillus anthracis and Staphylococcus aureus. PGN activated the human DCs, as judged by the increased expression of surface HLA-DR, CD83, the T cell costimulatory molecules CD40 and CD86, and the chemokine receptor CCR7. PGN elicited the DC production of IL-23, IL-6, and IL-1β but not IL-12p70. The PGN-stimulated DCs induced the differentiation of naïve allogeneic CD4+ T cells into T helper (TH) cells producing IL-17 and IL-21. Notably, the DCs from a subset of donors did not produce significant levels of IL-23 and IL-1β upon PGN stimulation, suggesting that common polymorphisms in immune response genes regulate the PGN response. In sum, purified PGN is a highly stimulatory cell wall component that activates human DCs to secrete proinflammatory cytokines and promote the differentiation of TH17 cells that are important for neutrophil recruitment in extracellular bacterial infections.

Effects of Magnetic Nanoparticles on the Functional Activity of Human Monocytes and Dendritic Cells.

The use of nanoparticles in medicine is sometimes hampered by their potential to activate immune cells, eliciting inflammation or allergy. We investigated whether magnetic nanoparticles (MNPs) or biomimetic magnetic nanoparticles (BMNPs) affect relevant activities of human monocytes. We found that the nanoparticles neither elicited the production of pro-inflammatory mediators IL-6 and TNFα by resting monocytes (when BMNP dose < 300 μg/mL) nor enhanced their secretion induced by R848, a molecule engaging virus-recognizing receptors, or bacterial lipopolysaccharide (LPS). MNPs and BMNPs neither induced the generation of reactive oxygen species (ROS), nor affected the ROS production elicited by the NADPH oxidase activator phorbol myristate acetate (PMA) or the fungal derivative β-glucan. BMNPs, but not MNPs, caused an up-regulation of the maturation markers CD80, CD83, and CD86 in immature monocyte-derived dendritic cells (DCs), whereas both nanoparticles did not affect the LPS-induced expression of these markers. Moreover, the nanoparticles were greedily ingested by monocytes and DCs without altering their viability. Therefore, these nanoparticles are candidates for medical applications because they do not activate pro-inflammatory activities of monocytes. Furthermore, their ability to stimulate DC maturation could be used for the design of vaccines. Moreover, harmlessly engulfed nanoparticles could be vehicles to carry molecules inside the immune cells to regulate the immune response.

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models

We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B.

Fuzzy Model for the Automatic Recognition of Human Dendritic Cells

Background and objective: Nowadays, foodborne illness is considered one of the most outgrowing diseases in the world, and studies show that its rate increases sharply each year. Foodborne illness is considered a public health problem which is caused by numerous factors, such as food intoxications, allergies, intolerances, etc. Mycotoxin is one of the food contaminants which is caused by various species of molds (or fungi), which, in turn, causes intoxications that can be chronic or acute. Thus, even low concentrations of Mycotoxin have a severely harmful impact on human health. It is, therefore, necessary to develop an assessment tool for evaluating their impact on the immune response. Recently, researchers have approved a new method of investigation using human dendritic cells, yet the analysis of the geometric properties of these cells is still visual. Moreover, this type of analysis is subjective, time-consuming, and difficult to perform manually. In this paper, we address the automation of this evaluation using image-processing techniques. Methods: Automatic classification approaches of microscopic dendritic cell images are developed to provide a fast and objective evaluation. The first proposed classifier is based on support vector machines (SVM) and Fisher's linear discriminant analysis (FLD) method. The FLD-SVM classifier does not provide satisfactory results due to the significant confusion between the inhibited cells on one hand, and the other two cell types (mature and immature) on the other hand. Then, another strategy was suggested to enhance dendritic cell recognition results that are emitted from microscopic images. This strategy is mainly based on fuzzy logic which allows us to consider the uncertainties and inaccuracies of the given data. Results: These proposed methods are tested on a real dataset consisting of 421 images of microscopic dendritic cells, where the fuzzy classification scheme efficiently improved the classification results by successfully classifying 96.77% of the dendritic cells. Conclusions: The fuzzy classification-based tools provide cell maturity and inhibition rates which help biologists evaluate severe health impacts caused by food contaminants.

Conventional dendritic cells type 1 are strongly enriched, quiescent and relatively tolerogenic in local inflammatory arthritis.

Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about the functional specialization of human DC subsets in (local) inflammatory conditions. We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from a local inflammatory site, namely synovial fluid (SF) from patients suffering from a chronic inflammatory condition, Juvenile Idiopathic Arthritis (JIA) as well as patients with rheumatoid arthritis (RA). Paired PB and SF samples from 32 JIA and 4 RA patients were collected for mononuclear cell isolation. Flow cytometry was done for definition of antigen presenting cell (APC) subsets. Cell sorting was done on the FACSAria II or III. RNA sequencing was done on SF APC subsets. Proliferation assays were done on co-cultures after CD3 magnetic activated cell sorting (MACS). APC Toll-like receptor (TLR) stimulation was done using Pam3CSK4, Poly(I:C), LPS, CpG-A and R848. Cytokine production was measured by Luminex. cDC1, a relatively small DC subset in blood, are strongly enriched in SF, and showed a quiescent immune signature without a clear inflammatory profile, low expression of pathogen recognition receptors (PRRs), chemokine and cytokine receptors, and poor induction of T cell proliferation and cytokine production, but selective production of IFNλ upon polyinosinic:polycytidylic acid exposure. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including IL-17. Although the cDC2 and monocytes showed an overlapping transcriptional core profile, there were clear differences in the transcriptional landscape and functional features, indicating that these cell types retain their lineage identity in chronic inflammatory conditions. Our findings suggest that at the site of inflammation, there is specific functional programming of human DCs, especially cDC2. In contrast, the enriched cDC1 remain relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a potentially more regulatory role.

Diurnal changes and topographical distribution of ocular surface epithelial dendritic cells in humans, and repeatability of density and morphology assessment.

Dendritic cells (DC) play a crucial role in ocular surface defence. DC can be visualised in vivo by confocal microscopy but have not yet been fully characterised in humans. This study investigated the diurnal variation, topographical distribution and repeatability of DC density and morphology measurements. In vivo confocal microscopy (IVCM) was conducted on 20 healthy participants (mean age 32.7± 6.4 years, 50% female) at baseline and repeated after 30 minutes, 2, 6 and 24 h. Images were captured at the corneal centre, inferior whorl, corneal periphery, limbus and bulbar conjunctiva. DC were counted manually, and their morphology was assessed for cell body size, presence of dendrites, and presence of long and thick dendrites. Mixed-model analysis, non-parametric analyses, Bland and Altman plots, coefficient of repeatability (CoR) and kappa were used. There were no significant changes in DC density (p≥ 0.74) or morphology (p> 0.07) at any location over the 24-h period. The highest DC density was observed at the corneal limbus followed by the peripheral cornea (p< 0.001), with the lowest density at the corneal centre, inferior whorl and bulbar conjunctiva. Most DC at the corneal periphery, limbus and bulbar conjunctiva had larger cell bodies compared with the corneal centre (p≤ 0.01), and the presence of long dendrites was observed mostly at non-central locations. Day-to-day CoR for DC density ranged from ±28.1 cells/mm2 at the corneal centre to ±56.4 cells/mm2 at the limbus. Day-to-day agreement of DC morphology determined by kappa ranged from 0.5 to 0.95 for cell body size, 0.60 to 0.95 for presence of dendrites, and 0.55 to 0.80 for the presence of long dendrites at various locations. No diurnal changes are apparent in corneal or conjunctival DC. Substantial topographical differences exist in DC density and morphology. IVCM provides good repeatability of DC density and acceptable agreement of DC morphology.

Study and isolation of human intestinal dendritic cell and macrophage subsets.

Dendritic cells and macrophages are the main antigen-presenting cells (APC). In the gut, they control the mechanisms of tolerance toward commensals and nutrients, at the time that they maintain their capacity to trigger immune responses against invading pathogens. Nevertheless, this balance is not perfect as it can get disrupted like in inflammatory bowel disease (where they drive an abnormal immune response against the microbiota) or in coeliac disease (where they trigger an immune response against dietary gluten). Therefore, the study of human intestinal APC subsets is crucial not just to get a deeper insight in the mechanisms of human intestinal homeostasis, but also to understand the pathogenesis of inflammatory bowel disease and coeliac disease. Nevertheless, their study is quite complicated as despite their relevance, their numbers are scare in the intestinal mucosa. Therefore, we hereby describe different approaches to study human intestinal dendritic cell and macrophage subsets in the human intestinal mucosa.