Distinct Dendritic Cells Subsets Research Articles

Expression profile of novel cell surface molecules on different subsets of human peripheral blood antigen‐presenting cells

Although major steps have been recently made in understanding the role of the distinct subsets of dendritic cells (DC)/antigen-presenting cells (APC), further studies are required to unravel their precise role, including in-depth immunophenotypic characterisation of these cells. Here, we used eight-colour flow cytometry to investigate the reactivity of a panel of 72 monoclonal antibodies (including those clustered in seven new Cluster of Differentiation, CD) on different subsets of APC in peripheral blood (PB) samples from five healthy adults. These experiments were performed in the context of the Tenth International Workshop on Human Leukocyte Differentiation Antigens (HLDA10). Plasmacytoid DC was the only cell population that expressed CD85g and CD195, whereas they lacked all of the other molecules investigated. In contrast, myeloid DC mostly expressed inhibitory C-type lectin receptors (CLRs) and other inhibitory-associated molecules, whereas monocytes expressed both inhibitory and activating CLRs, together with other phagocytosis-associated receptors. Within monocytes, progressively lower levels of expression were generally observed from classical monocytes (cMo) to SLAN− and SLAN+ non-classical monocytes (ncMo) for most of the molecules expressed, except for the CD368 endocytic receptor. This molecule was found to be positive only in cMo, and the CD369 and CD371 modulating/signalling receptors. In addition, the CD101 inhibitory molecule was found to be expressed at higher levels in SLAN+ vs SLAN− ncMo. In summary, the pattern of expression of the different signalling molecules and receptors analysed in this work varies among the distinct subsets of PB APCs, with similar profiles for molecules within each functional group. These findings suggest unique pattern-recognition and signalling capabilities for distinct subpopulations of APCs, and therefore, diverse functional roles.

Differential Intrasplenic Migration of Dendritic Cell Subsets Tailors Adaptive Immunity

SUMMARYEvidence suggests that distinct splenic dendritic cell (DC) subsets activate either CD4+ or CD8+ T cells in vivo. This bias has been partially ascribed to differential antigen presentation; however, all DC subsets can activate both T cell lineages in vitro. Therefore, we tested whether the organization of DC and T cell subsets in the spleen dictated this preference. We discovered that CD4+ and CD8+ T cells segregated within splenic T cell zones prior to immunization. After intravenous immunization, the two major conventional DC populations, distinguished by 33D1 and XCR1 staining, migrated into separate regions of the T cell zone: 33D1+ DCs migrated into the CD4+ T cell area, whereas XCR1+ DCs migrated into the CD8+ T cell area. Thus, the post-immunization location of each DC subset correlated with the T cell line-age it preferentially primes. Preventing this co-localization selectively impaired either CD4+ or CD8+ T cell immunity to blood-borne antigens.

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.

Dendritic Cell Subsets in the Skin and Their Functional Role in Contact Hypersensitivity

Objectives: Skin is a primary epithelial organ, which protects our body from the surrounding environment. Besides physical barrier machineries, skin also contains a number of immune components that actively participate in the protective immune responses. Dendritic cells (DCs) are professional antigen-presenting cells and they are essentially required for mediating innate and adaptive immunity. Although emerging studies have demonstrated distinct resident DC subsets in the skin, their subset-specific functions still need to be elucidated. Methods: We reviewed recent works on the DC subset discrimination and specification in the mouse skin. To understand the DC subset-dependent functional diversity, we especially focused on the murine contact hypersensitivity (CHS), an experimental model of human allergic contact dermatitis. Furthermore, we discussed our recent work on the role of epidermal Langerhans cells (LCs) in CHS. Results: Murine skin harbors at least three DC subsets: (i) epidermal LCs, (ii) Langerin+ dermal DCs and (iii) Langerin- dermal DCs. Using more sophisticated cell markers, recent study has described monocyte-derived DCs in the skin. The role of each DC subset in CHS was somewhat inconsistent and redundant from study to study and needs further elaborative works for the general acceptance. Conclusion: Continuous efforts to understand the functional diversity among each cutaneous DC subset will be needed to develop the new anti-inflammatory and anti-tumor strategies by targeting relevant skin DC population in vivo.

TIM-4 is differentially expressed in the distinct subsets of dendritic cells in skin and skin-draining lymph nodes and controls skin Langerhans cell homeostasis.

T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on dendritic cells (DC) and macrophages, plays an essential role in regulating immune responses. Langerhans cells (LC), which are the sole DC subpopulation residing at the epidermis, are potent mediators of immune surveillance and tolerance. However, the significance of TIM-4 on epidermal LCs, along with other cutaneous DCs, remains totally unexplored. For the first time, we discovered that epidermal LCs expressed TIM-4 and displayed an increased level of TIM-4 expression upon migration. We also found that dermal CD207+ DCs and lymph node (LN) resident CD207−CD4+ DCs highly expressed TIM-4, while dermal CD207− DCs and LN CD207−CD4− DCs had limited TIM-4 expressions. Using TIM-4-deficient mice, we further demonstrated that loss of TIM-4 significantly upregulated the frequencies of epidermal LCs and LN resident CD207−CD4+ DCs. In spite of this, the epidermal LCs of TIM-4-deficient mice displayed normal phagocytic and migratory abilities, comparable maturation status upon the stimulation as well as normal repopulation under the inflamed state. Moreover, lack of TIM-4 did not affect dinitrofluorobenzene-induced contact hypersensitivity response. In conclusion, our results indicated that TIM-4 was differentially expressed in the distinct subsets of DCs in skin and skin-draining LNs, and specifically regulated epidermal LC and LN CD207−CD4+ DC homeostasis.

A dendritic cell subset designed for oral tolerance.

Studies using genetic tools have identified the distinct dendritic cell subsets that ensure tolerance to oral antigens in the antigen-rich environment of the gut and suggest a 'division of labor' for protective immunity.

Effects of Dendritic Cell Subset Manipulation on Airway Allergy in a Mouse Model

Background: Two major distinct subsets of dendritic cells (DCs) are arranged to regulate immune responses: DEC-205+ DCs drive Th1 polarization and 33D1+ DCs establish Th2 dominancy. Th1 polarization can be achieved either by depletion of 33D1+ DCs with a 33D1-specific monoclonal antibody (mAb) or by activation of DEC-205+ DCs via intraperitoneal injection of α-galactosylceramide (α-GalCer). We studied the effect of 33D1+ DC depletion or DEC-205+ DC activation in vivo using an established mouse model of allergic rhinitis (AR). Methods: Mice were injected intraperitoneally with OVA plus alum and challenged 4 times with daily intranasal administration of OVA. Immediately after the last challenge, allergic symptoms such as sneezing and nasal rubbing as well as the number of cells in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF) were counted. The levels of serum OVA-specific IgG1, IgG2a, and IgE were also determined by ELISA. Results: The allergic symptom scores were significantly decreased in 33D1+ DC-depleted or DEC-205+ DC-activated AR mice. The levels of OVA-specific IgG1, IgG2a, and IgE, and the number of NALF cells, but not BALF cells, were reduced in 33D1+ DC-depleted but not in DEC-205+ DC-activated AR mice. Moreover, the activated DEC-205+ DCs suppressed histamine release from IgE-sensitized mast cells, probably through IL-12 secretion. Conclusions: The manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing histamine release from IgE-sensitized mast cells by driving the immune response towards Th1 dominancy via activation of DEC-205+ DCs in vivo.

Elevation of HLA-G-expressing DC-10 cells in patients with gastric cancer

DC-10 is a distinct subset of human tolerogenic dendritic cells (DCs) which express high levels of human leukocyte antigen-G (HLA-G). DC-10 could induce adaptive type 1 regulatory T cells through the IL-10 dependent ILT4/HLA-G signaling pathway. However, the significance of DC-10 in malignancies remains unclear. In this study, the frequency and mean fluorescence intensity (MFI) of HLA-G+ DC-10 in the peripheral blood of 124 patients with gastric cancer (GC) and 130 normal controls was analyzed with flow cytometry. Plasma sHLA-G was analyzed with ELISA. Results showed both the percentages of peripheral HLA-G+ DC-10 (median: 0.13% vs 0.01%; p<0.01) and MFI of HLA-G on these cells (median: 310.0 vs 91.5; p<0.01) were dramatically increased in GC patients than in normal controls. The frequency of HLA-G+ DC-10 in GC patients was strongly relative to the tumor grade (p=0.021). sHLA-G levels in GC patients were significantly higher than in healthy controls (median: 85.80U/ml vs 61.20U/ml; p<0.01). There was no significant correlation between the percentage of DC-10 and plasma sHLA-G (p>0.05). However, the increased HLA-G+ DC-10, HLA-G MFI and plasma sHLA-G in patients with gastric cancer could be a diagnostic factor with the area under the ROC curve with 0.947 (p<0.01), 0.882 (p<0.01) and 0.700 (p<0.01) respectively. Given the immune tolerant function of DC-10 could play, the increased DC-10 might play an important role in immune suppression for patients with gastric cancer, while more studies are necessary to illustrate the clinical relevance of DC-10 in cancer patients.

The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents.

Dendritic cells (DCs) form a complex network of cells that initiate and orchestrate immune responses against a vast array of pathogenic challenges. Developmentally and functionally distinct DC subtypes differentially regulate T-cell function. Importantly it is the ability of DC to capture and process antigen, whether from pathogens, vaccines, or self-components, and present it to naive T cells that is the key to their ability to initiate an immune response. Our typical isolation procedure for DC from murine spleen was designed to efficiently extract all DC subtypes, without bias and without alteration to their in vivo phenotype, and involves a short collagenase digestion of the tissue, followed by selection for cells of light density and finally negative selection for DC. The isolation procedure can accommodate DC numbers that have been artificially increased via administration of fms-like tyrosine kinase 3 ligand (Flt3L), either directly through a series of subcutaneous injections or by seeding with an Flt3L secreting murine melanoma. Flt3L may also be added to bone marrow cultures to produce large numbers of in vitro equivalents of the spleen DC subsets. Total DC, or their subsets, may be further purified using immunofluorescent labeling and flow cytometric cell sorting. Cell sorting may be completely bypassed by separating DC subsets using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Our procedure enables efficient separation of the distinct DC subsets, even in cases where mouse numbers or flow cytometric cell sorting time is limiting.

Dendritic Cells and Their Multiple Roles during Malaria Infection.

Dendritic cells (DCs) play a central role in the initiation of adaptive immune responses, efficiently presenting antigens to T cells. This ability relies on the presence of numerous surface and intracellular receptors capable of sensing microbial components as well as inflammation and on a very efficient machinery for antigen presentation. In this way, DCs sense the presence of a myriad of pathogens, including Plasmodium spp., the causative agent of malaria. Despite many efforts to control this infection, malaria is still responsible for high rates of morbidity and mortality. Different groups have shown that DCs act during Plasmodium infection, and data suggest that the phenotypically distinct DCs subsets are key factors in the regulation of immunity during infection. In this review, we will discuss the importance of DCs for the induction of immunity against the different stages of Plasmodium, the outcomes of DCs activation, and also what is currently known about Plasmodium components that trigger such activation.

SlanDCs/M-DC8+ cells constitute a distinct subset of dendritic cells in human tonsils [corrected

Human blood dendritic cells (DCs) include three main distinct subsets, namely the CD1c+ and CD141+ myeloid DCs (mDCs) and the CD303+ plasmacytoid DCs (pDCs). More recently, a population of slan/M-DC8+ cells, also known as "slanDCs", has been described in blood and detected even in inflamed secondary lymphoid organs and non-lymphoid tissues. Nevertheless, hallmarks of slan/M-DC8+ cells in tissues are poorly defined. Herein, we report a detailed characterization of the phenotype and function of slan/M-DC8+ cells present in human tonsils. We found that tonsil slan/M-DC8+ cells represent a unique DC cell population, distinct from their circulating counterpart and also from all other tonsil DC and monocyte/macrophage subsets. Phenotypically, slan/M-DC8+ cells in tonsils display a CD11c+HLA-DR+CD14+CD11bdim/negCD16dim/negCX3CR1dim/neg marker repertoire, while functionally they exhibit an efficient antigen presentation capacity and a constitutive secretion of TNFα. Notably, such DC phenotype and functions are substantially reproduced by culturing blood slan/M-DC8+ cells in tonsil-derived conditioned medium (TDCM), further supporting the hypothesis of a full DC-like differentiation program occurring within the tonsil microenvironment. Taken together, our data suggest that blood slan/M-DC8+ cells are immediate precursors of a previously unrecognizedcompetent DC subset in tonsils, and pave the way for further characterization of slan/M-DC8+ cells in other tissues.

Profiling of primary peripheral blood- and monocyte-derived dendritic cells using monoclonal antibodies from the HLDA10 Workshop in Wollongong, Australia.

Dendritic cells (DCs) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes. Mainly three phenotypically and functionally distinct DC subsets are described in the human peripheral blood (PB): plasmacytoid DCs (pDCs), which express the key marker CD303 (BDCA-2), and two myeloid DC subsets (CD1c+ DC (mDC1) and CD141+ DC (mDC2)), which express the key markers CD1c (BDCA-1) and CD141 (BDCA-3), respectively. In addition to these primary cell subsets, DCs can also be generated in vitro from either CD34+ stem/progenitor cells in the presence of Flt3 (Fms-related tyrosine kinase 3) ligand or from CD14+ monocytes (monocyte-derived DCs (mo-DCs)) in the presence of granulocyte–macrophage colony-stimulating factor+interleukin-4 (GM-CSF+IL-4). Here we compare the reactivity patterns of HLDA10 antibodies (monoclonal antibody (mAb)) with pDCs, CD1c+ DCs and CD141+ DCs, as well as with CD14+-derived mo-DCs cultured for 7 days in the presence of 100 ng/ml GM-CSF plus 20 ng/ml IL-4. A detailed profiling of these DC subsets based on immunophenotyping and multicolour flow cytometry analysis is presented. Using the panel of HLDA10 Workshop mAb, we could verify known targets selectively expressed on discrete DC subsets including CD370 as a selective marker for CD141+ DCs and CD366 as a marker for both myeloid subsets. In addition, vimentin and other markers are heterogeneously expressed on all three subsets, suggesting the existence of so far not identified DC subsets.

Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses.

Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving “undruggable” targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205+ DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR.

Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.

ID: 244 : Eosinophil IL-13 polarizes CD11b+ CD103+ dendritic cells to initiate Th2 immune responses in the gut associated lymphoid tissue

Dendritic cells are key to the initiation and regulation of immune responses. In the gut there are three distinct dendritic cell (DC) subsets, defined by CD11b and CD103, each with different developmental requirements and distinct functional potential. Recent evidence has described a role for the different gut DC subsets in the generation of Th17 and Treg responses, but little is yet known of their contribution to the initiation of Th2 immune responses in the intestines. We used the nematode Heligmosomoides polygyrus to initiate a potent Th2 response in the small intestine. We found that during H. polygyrus infection CD11b+ CD103+ DC preferentially migrated from the site of infection to the local lymph node. These DC showed increased expression of PDL2 and had an enhanced capacity to prime naive CD4+ T cell toward Th2. Migration and activation of CD11b+ CD103+ DC was dependent on the cytokine IL-13, which was secreted by activated eosinophils at the site of infection. Together our data suggest that the initiation of a Th2 response in the intestines involves early activation of eosinophils, which secrete the cytokine needed to activate and mobilize Th2-driving DCs.

Spatiotemporally Distinct Interactions with Dendritic Cell Subsets Facilitates CD4+ and CD8+ T Cell Activation to Localized Viral Infection

The dynamics of when and where CD4(+) Tcells provide help for CD8(+) Tcell priming and which dendritic cells (DCs) activate CD4(+) Tcells invivo after localized infection are poorly understood. By using a cutaneous herpes simplex virus infection model combined with intravital 2-photon imaging of the draining lymph node (LN) to concurrently visualize pathogen-specific CD4(+) and CD8(+) Tcells, we found that early priming of CD4(+) Tcells involved clustering with migratory skin DCs. CD8(+) Tcells did not interact with migratory DCs and their activation was delayed, requiring later clustering interactions with LN-resident XCR1(+) DCs. CD4(+) Tcells interacted with these late CD8(+) Tcell clusters on resident XCR1(+) DCs. Together, these data reveal asynchronous Tcell activation by distinct DC subsets and highlight the key role of XCR1(+) DCs as the central platform for cytotoxic T lymphocyte activation and the delivery of CD4(+) Tcell help.

Human dendritic cell subsets and function in health and disease.

The method of choice for the development of new vaccines is to target distinct dendritic cell subsets with antigen in vivo and to harness their function in situ to enhance cell-mediated immunity or induce tolerance to specific antigens. The innate functions of dendritic cells themselves may also be targeted by inhibitors or activators that would target a specific function such as interferon production, potentially important in autoimmune disease and chronic viral infections. Importantly targeting dendritic cells requires detailed knowledge of both the surface phenotype and function of each dendritic cell subset, including how they may respond to different types of vaccine adjuvants, their ability to produce soluble mediators and to process and present antigens and induce priming of naïve T cells. This review summarizes our knowledge of the functional attributes of the human dendritic cell subsets in the steady state and upon activation and their roles in human disease.

Intestinal CD103(+)CD11b(-) dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells.

A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A–diphtheria toxin receptor (DTR) mice after depletion of CD103+CD11b− DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103+CD11b+ DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103+CD11b− DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-γ (IFN-γ)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103+CD11b− DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-γ that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin.

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin+ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin+ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.

Modular expression analysis reveals functional conservation between human Langerhans cells and mouse cross-priming dendritic cells.

Characterization of functionally distinct dendritic cell (DC) subsets in mice has fueled interest in whether analogous counterparts exist in humans. Transcriptional modules of coordinately expressed genes were used for defining shared functions between the species. Comparing modules derived from four human skin DC subsets and modules derived from the Immunological Genome Project database for all mouse DC subsets revealed that human Langerhans cells (LCs) and the mouse XCR1(+)CD8α(+)CD103(+) DCs shared the class I-mediated antigen processing and cross-presentation transcriptional modules that were not seen in mouse LCs. Furthermore, human LCs were enriched in a transcriptional signature specific to the blood cross-presenting CD141/BDCA-3(+) DCs, the proposed equivalent to mouse CD8α(+) DCs. Consistent with our analysis, LCs were highly adept at inducing primary CTL responses. Thus, our study suggests that the function of LCs may not be conserved between mouse and human and supports human LCs as an especially relevant therapeutic target.