Characterization Of Dendritic Cells Research Articles

Abstract 3001: Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma

Abstract WHO grade 4 glioblastoma multiforme (GBM) has a very poor prognosis even after standard treatment with surgery, radiotherapy, and chemotherapy. Tumor recurrence is virtually inevitable when it is fatal. Chimeric Antigen Receptor (CAR)-T cell therapy is a promising new modality of therapy for GBM patients. Even though second-generation CAR-T cells may be considered to operate autonomously because co-stimulation is inherent to CAR design, the tumor microenvironment of GBM is particularly hostile to immune cells and their normal functions. In our recently published work (Gargett et al. J Immunother Cancer 2022; 10(9): e005187), CAR-T cells specific for the glioblastoma antigen, GD2, had improved antitumor activity if the CAR-T cells co-expressed IL-15. Although IL-15 co-expression prolonged control of intracranial tumor xenografts, tumors escaped immune control in most cases. Our early data suggest that a relative lack of intratumoral dendritic cells (DCs) may be a contributory factor. DCs are central to the initiation and maintenance of adaptive immune responses. Hence, we were interested to survey and characterize the DC content of glioblastoma tissues obtained either from human surgical samples or mice bearing orthotopic xenografts and syngrafts. We have used multi-colour flow cytometry to enumerate and evaluate the maturation status of subsets of DCs together with other types of immune cells among single cell suspensions of isolated glioblastoma tissues. Our preliminary findings are: Total DCs (mean +/- SD), which were defined as lineage-negative and HLA-DR (+), were significantly reduced among total viable cells of glioblastoma tissues (0.845% +/- 0.569) and peripheral blood mononuclear cells (PBMCs) (0.755% +/- 0.601) from GBM patients (n=12) compared to PBMCs (1.77% +/- 0.611) from healthy donors (n=12).No significant differences were detected in the percentages of total DCs among splenocytes of nontumor-bearing C57BL/6 (B6) mice (n=6) compared to B6 bearing othotopic syngrafts of the GL261 (n=3) or C2TA (n=3) glioblastoma cell lines. We will present more detailed analyses from human glioblastoma samples. In addition, we will present a more extensive characterization of DCs from the orthotopic murine models of glioblastoma: we will compare DCs and other types of immune cells in tumor and in the primary and secondary lymphoid tissues of both tumor-bearing and nontumor-bearing mice. We expect that the final data will fill a current gap in knowledge and indicate possible therapeutic strategies that might correct for the quantitative and qualitative defects in dendritic cells in glioblastoma. We hypothesize that adequate correction of these defects in vivo may augment the anti-glioblastoma activity of CAR-T cell therapies. Citation Format: Michael P. Brown, Tessa Gargett, Bryan J. Gardam. Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3001.

Dendritic cells and their associated pro-inflammatory cytokines augment to the inflammatory milieu in vitiligo skin

Vitiligo is a progressive, autoimmune, hypomelanotic acquired disorder of skin which is characterized by depigmentation. The initial immunological events of this diseases are still at enigma that includes breach of immune tolerance, and defect in antigen presentation. Hence, we aimed to explore role of Dendritic cells (DCs) and its associated cytokines in the pathogenesis of generalized vitiligo (GV) patients. For this case-control study, 20 active patients and controls were enrolled. Phenotypic characterization of myeloid and plasmacytoid DCs (mDCs, pDCs) were done by flow-cytometry. Primary culture of DCs was done by monocyte differentiation supplemented with rIL-4 and rGM-CSF. Functional analysis DCs related cytokines and co-stimulatory molecules (CD80, CD40) was done by ELISA and qPCR respectively. Tissue localization of DCs was evaluated by immunohistochemistry. The frequency of mDCs (0.3715% v/s 0.188%) and pDCs (0.2331% v/s 0.1156%) were elevated in patients as compared to controls. Circulatory level of IL-12, TNF-α were significantly higher whereas IFN-α was decreased in patients than controls. Similar results were obtained in the culture supernatants of patients. Relative mRNA expression profiling of co-stimulatory molecules (CD80, CD40) were found to be up regulated in patient's skin. Tissue localization of Langerhans cells (Langerin, CD1a+) were found to be significantly higher in patients. Elevated frequency of mDCs and pDCs along with elevated levels of IL-12, TNF-α and CD80, CD40 may contribute in defective antigen presentation of DCs. Altered pro-inflammatory and anti-inflammatory cytokines along with tissue localization of Langerhans cells might be involved in the pathogenesis of GV. These DCs associated cytokines can be explored as a therapeutic target in future.

FMS-like tyrosine kinase 3 ligand (FLT3L) administration alters rat classical and plasmacytoid dendritic cell phenotype in a tissue-specific manner

Abstract Background: Characterization of tissue-resident dendritic cell (DC) subsets, including classical DCs (cDCs) and plasmacytoid DCs (pDCs), is complicated experimentally by their rare nature. Many investigators have artificially increased DC yields through administration of the cytokine FMS-like tyrosine kinase 3 ligand (FLT3L). Despite its previous use experimentally, no study has yet described the effects of in vivo FLT3L administration on rat tissue-resident DCs. This study provides an advanced characterization of rat tissue-resident cDCs and pDCs following FLT3L injection. Methods: Lewis rats were injected once daily for 10 days with PBS vehicle or with 10, 50, or 100μg of FLT3L. Animals were sacrificed on the 11th day and dissected to obtain liver, spleen, and mesenteric lymph nodes. Cells were stained for 13-color flow cytometry and data was acquired on the Cytek Aurora spectral cytometer. Results: cDC and pDC enumeration increased in a dose-dependent manner following FLT3L administration. Expression levels of MHC I, MHC II, CD40, CD80, CD86, and PD-L1 were evaluated on liver, spleen, and mesenteric lymph node-resident cells. MHC II, CD80, and CD86 levels were significantly decreased on liver-resident cDCs but remained unchanged or increased on spleen and lymph node cDCs. Changes in MHC I/II and costimulatory molecule expression levels on pDCs varied by tissue and marker. Conclusions: FLT3L administration in vivo increased cDC and pDC enumeration, generated immature cDCs in the liver, and increased cDC maturation in the spleen and mesenteric lymph nodes. This comprehensive characterization of rat liver, spleen, and lymph node-resident DCs is key to understanding the effects of FLT3L on cDC and pDC phenotype.

The Prevalence of Gingival Dendritic Cell Subsets in Periodontal Patients

As the most potent cells activating and polarizing naive T cells, dendritic cells (DCs) are of major importance in the induction of immunity and tolerance. DCs are a heterogeneous population of antigen-presenting cells that are widely distributed in lymphoid and nonlymphoid tissues. Murine studies have highlighted the important role of oral DCs and Langerhans cells (LCs) in orchestrating the physiological homeostasis of the oral mucosa. DCs are also critically involved in pathological conditions such as periodontal diseases, in which gingival DCs appear to have special localization and function. While the characterization of human DCs in health and disease has been extensively investigated in various tissues, this topic was rarely studied in human gingiva. Here, we employed an up-to-date approach to characterize by flow cytometry the gingival DCs of 27 healthy subjects and 21 periodontal patients. Four distinct subsets of mononuclear phagocytes were identified in healthy gingiva: conventional DC type 1 (cDC1), cDC2, plasmacytoid DCs (pDCs), and LCs. In periodontitis patients, the frequencies of gingival LCs and pDCs were dysregulated, as LCs decreased, whereas pDCs increased in the diseased gingiva. This shift in the prevalence of DCs was accompanied by increased expression of the proinflammatory cytokines interleukin (IL)–1β, interferon (IFN)–α, and IFN-γ, while the anti-inflammatory cytokine IL-10 was suppressed. We further found that smoking, a known risk factor of periodontitis, specifically reduces gingival LCs in healthy individuals, indicating a possible role of LCs in the elevated severity of periodontitis in smokers. Collectively, this work reveals the various DC subsets residing in the human gingiva and the impact of periodontitis, as well as smoking, on the prevalence of each subset. Our findings provide a foundation toward understanding the role of human DCs in orchestrating physiological oral immunity and set the stage for the evaluation and modulation of shifts in immunity associated with periodontitis.

Comprehensive Phenotyping of Dendritic Cells in Cancer Patients by Flow Cytometry.

Dendritic cells (DCs) play a crucial role in the complex interplay between tumor cells and the immune system. During the elimination phase of cancer immunoediting, immunostimulatory DCs are critical for the control of tumor growth. During the escape phase, regulatory DCs sustain tumor tolerance and contribute to the development of the immunosuppressive tumor microenvironment that characterizes this phase. Moreover, increasing evidence indicates that DCs are also critical for the success of cancer immunotherapy. Hence, there is increasing need to fully characterize DC subsets and their activatory/inhibitory profile in cancer patients. In this review, we describe the role played by different DC subsets in the different phases of cancer immunoediting, the function exerted by different activatory and inhibitory molecules expressed on DC surface, and the cytokines produced by distinct DC subsets, in order to provide an overview on the DC features that may be useful to be assessed when dealing with the flow cytometric characterization of DCs in cancer patients. © 2020 International Society for Advancement of Cytometry.

Development of Pig Conventional Dendritic Cells From Bone Marrow Hematopoietic Cells in vitro.

In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)-based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1+CD14–MHC-II+CD172a–/loCD1–CD163– DEC205+CD11R3loCD11R1+CD33+CD80/86+. They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4+CD8+ T cells, but were deficient in phagocyting inactivated Staphylococcus aureus (S. aureus). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1+CD14–MHC-II+C D172a+CD1+CD163–/loDEC205loCD11R3+CD11R1+CD33+CD80/86+; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4+CD8–/CD4+CD8+ T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14+ population was identified with the phenotype CADM1+CD14+MHC-II+CD172a+ CD1+CD163+DEC205–CD11R3+CD11R1+CD33–/loCD80/86+. They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14+ population was efficient in phagocyting S. aureus but showed less maturation upon TLR ligand stimulation than cDC1 or cDC2. The alternative methods of DC derivation including GM-CSF and/or IL-4 produced mostly CADM1– cells that did not fulfill the canonical phenotype of bona fide porcine DCs. Our study provides an exhaustive characterization of Flt3L-derived DCs with different methods that can help the in vitro study of the interaction of DCs with porcine-relevant pathogens.

Characterization of dendritic cells in human and experimental myocarditis.

AimsDendritic cells (DCs) are central mediators of adaptive immunity, and there is growing evidence of their role in myocardial inflammatory disease. We hypothesized that plasmacytoid and myeloid DCs are involved in the mechanisms of myocarditis and analysed these two main subtypes in human myocarditis subjects, as well as in a murine model of experimental autoimmune myocarditis (EAM).Methods and resultsCirculating DCs were analysed by flow cytometry in patients with acute myocarditis, dilated cardiomyopathy, and controls. Myocardial biopsies were immunostained for the presence of DCs and compared with non‐diseased controls. In a mouse model of acute myocarditis induced through synthetic cardiac myosine peptide injection, effects of immunomodulation including DC inhibition through MCS‐18 versus placebo treatment were tested at the peak of inflammation (Day 21), as well as 1 week later (partial recovery). Circulatory pDCs and mDCs were significantly reduced in myocarditis patients compared with controls (P < 0.01 for both) and remained so even after 6 months of follow‐up. Human myocarditis biopsies showed accumulation of pDCs (two‐fold CD304+/three‐fold CD123+, all P < 0.05) compared with controls. Myocardial pDCs and mDCs accumulated in EAM (P for both <0.0001). MCS‐18 treatment reduced pDC levels (P = 0.009), reduced myocardial inflammation (myocarditis score reduction from 2.6 to 1.8, P = 0.026), and improved ejection fraction (P = 0.03) in EAM at Day 21 (peak of inflammation). This effect was not observed during the partial recovery of inflammation on Day 28.ConclusionsCirculating DCs are reduced in human myocarditis and accumulate in the inflamed myocardium. MCS‐18 treatment reduces DCs in EAM, leading to amelioration of inflammation and left ventricular remodelling during the acute phase of myocarditis. Our data further elucidate the role of DCs and their specific subsets in acute inflammatory cardiomyopathies.

Characterization of dendritic cells and follicular dendritic cells in the hepatic lymph nodes and liver of sheep experimentally infected with Fasciola hepatica

Fasciola hepatica has been shown to have a high capacity for immunomodulation of the host response, making the development of protective vaccines extremely difficult. One of these immunomodulation mechanisms is the impairment of dendritic cells (DC) maturation and, therefore, suppression of antigenic presentation. The aim of this study was to evaluate the pathological changes as well as the characterization of two antigen presenting cells, DC (CD1b, CD83 and MHC-II positive) and follicular dendritic cells (FDC) (CNA.42, S100 and CD83 positive) by immunohistochemistry in the hepatic lymph nodes (HLN) and livers of sheep during the early stages of infection with F. hepatica [9 and 18 days post-infection (dpi)], compared with an uninfected group (UC) as a control. The results revealed a marked hyperplasia of HLN germinal centres at 9 and, in particular, 18 dpi, with respect to the UC group, with coincidental increased expression of CNA.42 in FDC of lymphoid follicles and CD1b in the DC of paracortical areas at 18 dpi. However, the expression of MHC-II and CD83 decreased at 9 and, particularly, at 18 dpi in HLN compared with that in the UC group. Since both markers are related to active presentation of antigens by DC and FDC, the results of the present study suggest that, despite the marked hyperplasia of HLN and increase in DC and FDC numbers during early stages of infection, the DC and FDC antigenic presentation capacity, as suggested by the expression of the markers MHC-II and CD83, is suppressed by the parasite. This suppression was not observed in the liver, probably because of the low number of DC. This is the first study of the immunophenotype of DCs and FDC in sheep infected with F. hepatica.

Characterization of dendritic cell (DC)-10 in recurrent miscarriage and recurrent implantation failure.

During pregnancy, the maternal immune system must tolerate the persistence of semi-allogeneic fetus in the maternal tissue. Inadequate recognition of fetal antigens may lead to pregnancy complications, such as recurrent miscarriage (RM) and recurrent implantation failure (RIF). Dendritic cells (DCs) are key regulators of protective immune responses and the development and maintenance of tolerance. Regarding that DCs are important in the establishment of immune tolerance in human pregnancy, it would be important to study the microenvironment in which DCs reside or are activated may affect their functions toward tolerance rather than active immune response. IL-10 plays a critical role in the maintenance of normal pregnancy, and the increased production of IL-10 is associated with successful pregnancy. In this study, we provide an in-depth comparison of the phenotype and cytokine production by DC-10 and other DC subsets, such as iDC and mDC. CD14+ monocyte-derived DCs were differentiated in the presence of IL-10 (DC-10) in vitro from ten normal fertile controls, six RM women and seven RIF women, and characterized for relevant markers. DC-10 was characterized by relatively low expression of costimulatory molecule CD86, as well as MHC class II molecule HLA-DR, high expression of tolerance molecules HLA-G, ILT2, ILT4 and immunosuppressive cytokine IL-10, but produced little or no proinflammatory cytokines, such as TNF-α, IL-6 and IL-12p70. Our study provides a better understanding of the phenotypical properties of DC-10, which may participate in the complex orchestration that leads to maternal immune tolerance and homeostatic environment in human pregnancy.

Costimulatory Molecules and Immune Checkpoints Are Differentially Expressed on Different Subsets of Dendritic Cells.

Dendritic cells (DCs) play a crucial role in initiating and shaping immune responses. The effects of DCs on adaptive immune responses depend partly on functional specialization of distinct DC subsets, and partly on the activation state of DCs, which is largely dictated by environmental signals. Fully activated immunostimulatory DCs express high levels of costimulatory molecules, produce pro-inflammatory cytokines, and stimulate T cell proliferation, whereas tolerogenic DCs express low levels of costimulatory molecules, produce immunomodulatory cytokines and impair T cell proliferation. Relevant to the increasing use of immune checkpoint blockade in cancer treatment, signals generated from inhibitory checkpoint molecules on DC surface may also contribute to the inhibitory properties of tolerogenic DCs. Yet, our knowledge on the expression of inhibitory molecules on human DC subsets is fragmentary. Therefore, in this study, we investigated the expression of three immune checkpoints on peripheral blood DC subsets, in basal conditions and upon exposure to pro-inflammatory and anti-inflammatory stimuli, by using a flow cytometric panel that allows a direct comparison of the activatory/inhibitory phenotype of DC-lineage and inflammatory DC subsets. We demonstrated that functionally distinct DC subsets are characterized by differential expression of activatory and inhibitory molecules, and that cDC1s in particular are endowed with a unique immune checkpoint repertoire characterized by high TIM-3 expression, scarce PD-L1 expression and lack of ILT2. Notably, this unique cDC1 repertoire was subverted in a group of patients with myelodysplastic syndromes included in the study. Applied to the characterization of DCs in the tumor microenvironment, this panel has the potential to provide valuable information to be used for investigating the role of DC subsets in cancer, guiding DC-targeting treatments, and possibly identifying predictive biomarkers for clinical response to cancer immunotherapy.

A Characterization of Dendritic Cells and Their Role in Immunotherapy in Glioblastoma: From Preclinical Studies to Clinical Trials

Glioblastoma (GBM) is the most common and fatal primary central nervous system malignancy in adults with a median survival of less than 15 months. Surgery, radiation, and chemotherapy are the standard of care and provide modest benefits in survival, but tumor recurrence is inevitable. The poor prognosis of GBM has made the development of novel therapies targeting GBM of paramount importance. Immunotherapy via dendritic cells (DCs) has garnered attention and research as a potential strategy to boost anti-tumor immunity in recent years. As the “professional” antigen processing and presenting cells, DCs play a key role in the initiation of anti-tumor immune responses. Pre-clinical studies in GBM have shown long-term tumor survival and immunological memory in murine models with stimulation of DC activity with various antigens and costimulatory molecules. Phase I and II clinical trials of DC vaccines in GBM have demonstrated some efficacy in improving the median overall survival with minimal to no toxicity with promising initial results from the first Phase III trial. However, there remains no standardization of vaccines in terms of which antigens are used to pulse DCs ex vivo, sites of DC injection, and optimal adjuvant therapies. Future work with DC vaccines aims to elucidate the efficacy of DC-based therapy alone or in combination with other immunotherapy adjuvants in additional Phase III trials.

Quantitative Proteomics Analysis Revealed Compromised Chicken Dendritic Cells Function at Early Stage of Very Virulent Infectious Bursal Disease Virus Infection.

Chicken dendritic cells (DCs) have been demonstrated to be susceptible to infectious bursal disease virus (IBDV), a causative agent of acute and immunosuppressed disease in young chicks known as infectious bursal disease. Further functional characterization of IBDV-infected DCs of chickens is required to provide a better understanding on the influence of the virus on chicken bone marrow-derived dendritic cells (BM-DCs) following very virulent (vv) IBDV infection. Membrane proteins of BM-DCs were extracted and the proteins were further denatured and reduced before performing labeling with isobaric tags for relative and absolute quantitation. The differential expression protein profiles were identified and quantified using liquid chromatography coupled with tandem mass spectrometry, and later validated using flow cytometry and real-time reverse transcriptase PCR. The analysis has identified 134 differentially regulated proteins from a total of 283 proteins (cutoff values of ≤0.67, ≥1.5, and ProtScore >1.3 at 95% confidence interval), which produced high-yield membrane fractions. The entry of vvIBDV into the plasma membrane of BM-DCs was observed at 3 hr postinfection by the disruption of several important protein molecule functions, namely apoptosis, RNA/DNA/protein synthesis, and transport and cellular organization, without the activation of proteins associated with signaling. At the later stage of infection, vvIBDV induced expression of several proteins, namely CD200 receptor 1-A, integrin alpha-5, HSP-90, cathepsin, lysosomal-associated membrane protein, and Ras-related proteins, which play crucial roles in signaling, apoptosis, stress response, and antigen processing as well as in secretion of danger-associated proteins. These findings collectively indicated that the chicken DCs are expressing various receptors regarded as potential targets for pathogen interaction during viral infection. Therefore, fundamental study of the interaction of DCs and IBDV will provide valuable information in understanding the role of professional antigen-presenting cells in chickens and their molecular interactions during IBDV infection and vaccination.

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

Dendritic cells and adipose tissue

Visceral adipose tissue inflammation in obesity is an established risk factor for metabolic syndrome, which can include insulin resistance, type 2 diabetes, hypertension and cardiovascular diseases. With obesity and related metabolic disorders reaching epidemic proportions globally, an understanding of the mechanisms of adipose tissue inflammation is crucial. Within the immune cell cohort, dendritic cells (DC) play a key role in balancing tolerance and immunity. Despite decades of research into the characterization of DC in lymphoid and non-lymphoid organs, their role in adipose tissue function is poorly understood. There is now an increasing interest in identification and characterization of DC in adipose tissue and understanding their function in regulating tissue metabolic homeostasis. This review provides an overview of the study of DC in adipose tissue, focusing on possible mechanisms by which DC may contribute to adipose tissue homeostasis.

The role of dendritic cells in male reproductive tract.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells. The central role of various DC subsets as bridges between innate and adaptive immunity has become more and more evident. However, the role of DC subsets in male reproductive tract remains largely unexplored, in particular distinct DC subsets (including myeloid and plasmacytoid DCs), their maturation stage, and tissue distribution, as well as state of health or disease. Furthermore, infection and inflammation of male genital tract are thought to be a primary etiological factor of male infertility. This review sheds some light on this complex and rapidly growing field. It summarized the recent findings and deals with the characterization and role of DCs in male reproductive tract, that is, testis, epididymis, prostate, seminal vesicle, semen, and foreskin, which might help to understand the immunopathological mechanisms of male infertility and design effective vaccines for male reproductive health.

Gut bacteria modulate dendritic cell priming of diabetogenic T cell effector functions in a mouse model of autoimmune diabetes

Abstract Increasing evidence implicates a contribution of the microbiome, within the gastrointestinal tract, in the initiation and progression of type 1 diabetes (T1D). Notably, our understanding of mechanisms whereby gut flora interaction with the host immune system can influence T1D pathogenesis remain poorly understood. Comparison of stool samples from the non-obese diabetic (NOD) mouse model of spontaneous T1D and non-obese resistant (NOR) mice revealed distinct bacterial flora communities present within the two strains correlated to a Th17 bias. Given that dendritic cells (DC) are critical in sampling luminal antigens and priming naïve T lymphocyte effector functions, we next interrogated the populations of CD11c+ DC within the lamina propria (LP) of NOD and NOR mice. Phenotypic characterization of LP DC revealed that NOR mice possessed more mature DC denoted by a higher frequency of CD11c+ MHCII+ cells and CD103+ CD86+ cells. We have previously shown that the oral administration of a gut bacterium Lactobaccillus johnsonii (LjN6.2), present in diabetes resistant rodents, was capable of conferring diabetes resistance to diabetes prone rodents while another strain Lactobacillus reuterii (LrTD1) had no effect. Notably, T1D resistance was correlated to a Th17 bias. Hence, we sought to investigate the influence of LjN6.2 on NOD and NOR DC in comparison to LrTD1 in vitro. We found that LjN6.2, but not LrTD1 mediated an increase in IL6 and IL10 cytokine production by DC. Together, these data suggest that gut bacteria have the important ability to shape the maturation and priming abilities of DC. These results could have critical implications regarding the ability of bacterial flora to regulate the generation of a diabetogenic immune repertoire.

TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells

Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M.scrofulaceum and we analyzed the effect on the activation of DCs. We found that M.scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M.scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M.scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M.scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M.scrofulaceum.

Characterization of dendritic cells in lip and oral cavity squamous cell carcinoma.

There may be differences in the antitumor immunity induced by dendritic cells (DCs) during the development of squamous cell carcinoma (SCC) located in the lip rather than in the oral cavity. The aim of this study was to evaluate the number of immature and mature DCs in SCC and potentially malignant disorders of the oral cavity and lip. Immunohistochemistry was used to identify the number (cells/mm(2) ) of immature (CD1a(+) ) or mature (CD83(+) ) DCs in samples of oral cavity SCC (OCSCC) (n = 39), lip SCC (LSCC) (n = 23), leukoplakia (LK) (n = 21), actinic cheilitis (AC) (n = 13), and normal mucosa of the oral cavity (OC control, n = 12) and the lip (lip control, n = 11). The number of CD1a(+) cells tended to be higher in the OC control samples compared with the LK (P = 0.04) and OCSCC (P = 0.21). Unlike, this cell population was lower in the lip control than in AC or LSCC (P < 0.05). The number of CD83(+) cells was increased in the LSCC samples compared with the AC and lip control (P = 0.0001) and in OCSCC compared with both the LK (P = 0.001) and OC control (P = 0.0001) samples. LSCC showed an elevated number of CD1a(+) and CD83(+) cells compared with OCSCC (P = 0.03). The population of mature DCs was lower than the population of immature DCs in all of the tested groups (P < 0.05). There were a greater number of both mature and immature DC populations in the LSCC samples than in the OCSCC, which could contribute to establishing a more effective immune antitumor response for this neoplasm.

Human monocyte-derived dendritic cells turn into foamy dendritic cells with IL-17A

Interleukin 17A (IL-17A) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases. In the field of immunometabolism, we have studied the impact of IL-17A on the lipid metabolism of human in vitro-generated monocyte-derived dendritic cells (DCs). Microarrays and lipidomic analysis revealed an intense remodeling of lipid metabolism induced by IL-17A in DCs. IL-17A increased 2-12 times the amounts of phospholipids, cholesterol, triglycerides, and cholesteryl esters in DCs. Palmitic (16:0), stearic (18:0), and oleic (18:ln-9c) acid were the main fatty acid chains present in DCs. They were strongly increased in response to IL-17A while their relative proportion remained unchanged. Capture of extracellular lipids was the major mechanism of lipid droplet accumulation, visualized by electron microscopy and Oil Red O staining. Besides this foamy phenotype, IL-17A induced a mixed macrophage-DC phenotype and expression of the nuclear receptor NR1H3/liver X receptor-α, previously identified in the context of atherosclerosis as the master regulator of cholesterol homeostasis in macrophages. These IL-17A-treated DCs were as competent as untreated DCs to stimulate allogeneic naive T-cell proliferation. Following this first characterization of lipid-rich DCs, we propose to call these IL-17A-dependent cells "foamy DCs" and discuss the possible existence of foamy DCs in atherosclerosis, a metabolic and inflammatory disorder involving IL-17A.