Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

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 nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. 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 coauthors, making it an essential resource for basic and clinical DC immunologists.

Restoration of dendritic cell homeostasis and Type I/Type III interferon levels in convalescent COVID-19 individuals.

BACKGROUND: Plasmacytoid and myeloid dendritic cells play a vital role in the protection against viral infections. In COVID-19, there is an impairment of dendritic cell (DC) function and interferon secretion which has been correlated with disease severity. RESULTS: In this study, we described the frequency of DC subsets and the plasma levels of Type I (IFNα, IFNß) and Type III Interferons (IFNλ1), IFNλ2) and IFNλ3) in seven groups of COVID-19 individuals, classified based on days since RT-PCR confirmation of SARS-CoV2 infection. Our data shows that the frequencies of pDC and mDC increase from Days 15-30 to Days 61-90 and plateau thereafter. Similarly, the levels of IFNα, IFNß, IFNλ1, IFNλ2 and IFNλ3 increase from Days 15-30 to Days 61-90 and plateau thereafter. COVID-19 patients with severe disease exhibit diminished frequencies of pDC and mDC and decreased levels of IFNα, IFNß, IFNλ1, IFNλ2 and IFNλ3. Finally, the percentages of DC subsets positively correlated with the levels of Type I and Type III IFNs. CONCLUSION: Thus, our study provides evidence of restoration of homeostatic levels in DC subset frequencies and circulating levels of Type I and Type III IFNs in convalescent COVID-19 individuals.

Unboxing dendritic cells: Tales of multi-faceted biology and function.

Often referred to as the bridge between innate and adaptive immunity, dendritic cells (DCs) are professional antigen-presenting cells (APCs) that constitute a unique, yet complex cell system. Among other APCs, DCs display the unique property of inducing protective immune responses against invading microbes, or cancer cells, while safeguarding the proper homeostatic equilibrium of the immune system and maintaining self-tolerance. Unsurprisingly, DCs play a role in many diseases such as autoimmunity, allergy, infectious disease and cancer. This makes them attractive but challenging targets for therapeutics. Since their initial discovery, research and understanding of DC biology have flourished. We now recognize the presence of multiple subsets of DCs distributed across tissues. Recent studies of phenotype and gene expression at the single cell level have identified heterogeneity even within the same DC type, supporting the idea that DCs have evolved to greatly expand the flexibility of the immune system to react appropriately to a wide range of threats. This review is meant to serve as a quick and robust guide to understand the basic divisions of DC subsets and their role in the immune system. Between mice and humans, there are some differences in how these subsets are identified and function, and we will point out specific distinctions as necessary. Throughout the text, we are using both fundamental and therapeutic lens to describe overlaps and distinctions and what this could mean for future research and therapies.

Altered Immune Regulation of Dendritic Cells and Enhanced Cytokine Production of T Cells in the Pathogenesis of Eosinophilic Chronic Rhinosinusitis.

INTRODUCTION: Eosinophilic chronic rhinosinusitis (ECRS) is a refractory chronic disease defined by recurrent nasal polyps with severe eosinophilic infiltration. This is mainly due to enhanced type 2-dominant immune responses, but the underlying mechanism is still not fully understood. OBJECTIVE AND METHODS: In the present study, we aimed to determine the characteristics of dendritic cells (DCs) and cytokine profiles of T cells in the peripheral blood of individuals with ECRS and age- and sex-matched healthy controls (HC). RESULTS AND CONCLUSION: The ratios of myeloid (m)DC1s to DCs and PD-L1+ mDC1s to mDC1s were higher in ECRS patients than in HC. The proportions of plasmacytoid (p)DCs in DCs, and human leukocyte antigen-DR+ pDCs and ILT3+ pDCs in pDCs were lower in ECRS patients than in HC. In a characterization of T cells, IL-4+CD4+, IFN-γ+CD4+, IL-4+IFN-γ+CD4+, IL-4+Foxp3+CD4+, IFN-γ+Foxp3+CD4+, IFN-γ+IL-4-Foxp3-CD4+, IL-4+CD8+, IL-4+IFN-γ+CD8+, and IL-4+Foxp3+CD8+ T-cell populations were significantly higher in ECRS patients than in HC. These results suggest that the enhanced immune regulation of mDC1, diminished capacity of pDCs, and increased proportion of the T-cell phenotypes in peripheral blood might be factors in ECRS pathogenesis.

Dendritic cells modulate c-kit expression on the edge between activation and death.

Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c-kit, the receptor for stem cell factor (SCF), nevertheless c-kit functional role and the regulation of its expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro-survival circuit, and that SCF increases phospho-AKT in c-kit+ mouse bone marrow-derived DCs (BMdDCs). Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c-kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI-/- mice showed that IFN-I pathway was required for c-kit down-regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c-kit mRNA expression. In agreement with the reduced c-kit levels, SCF pro-survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL-6 and IL-12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c-kit levels. Our studies suggest that c-kit down-modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.

Functional Role of Dendritic Cell Subsets in Cancer Progression and Clinical Implications.

Dendritic cells (DCs) constitute a complex network of cell subsets with common functions but also with many divergent aspects. All dendritic cell subsets share the ability to prime T cell response and to undergo a complex trafficking program related to their stage of maturation and function. For these reasons, dendritic cells are implicated in a large variety of both protective and detrimental immune responses, including a crucial role in promoting anti-tumor responses. Although cDC1s are the most potent subset in tumor antigen cross-presentation, they are not sufficient to induce full-strength anti-tumor cytotoxic T cell response and need close interaction and cooperativity with the other dendritic cell subsets, namely cDC2s and pDCs. This review will take into consideration different aspects of DC biology, including the functional role of dendritic cell subsets in both fostering and suppressing tumor growth, the mechanisms underlying their recruitment into the tumor microenvironment, as well as the prognostic value and the potentiality of dendritic cell therapeutic targeting. Understanding the specificity of dendritic cell subsets will allow to gain insights on role of these cells in pathological conditions and to design new selective promising therapeutic approaches.

Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.

BACKGROUND: Targeting angiogenesis has been and continues to be an attractive therapeutic modality in glioblastoma (GBM) patients. However, GBM rapidly becomes refractory to anti-VEGF therapies. Myeloid cell infiltration is an important determinant of tumor progression. Given that VEGF is a modulator of the innate immune response we sought to analyze the dynamics of this response in a mouse model of GBM undergoing anti-VEGF therapy. METHODS: We grafted GL261-DsRed cells in transgenic Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined recurrent spectral two-photon imaging with multiparametric cytometry, immunostaining, and brain clearing to characterize at two critical stages of tumor development (day 21 and day 28 after tumor grafting) the nature and spatial distribution of the innate response in control and bevacizumab-treated mice. RESULTS: We report that at an early stage (21 day), VEGF blockade has a detectable effect on the number of microglial cells but only a mild effect on the number of infiltrating myeloid cells. At a later stage (day 28), the treatment resulted in a specific adjustment of dendritic cell subsets. In treated mice, the number of monocytes and their monocyte-derived dendritic cells (moDC) progeny was increased by approximately twofold compared to untreated mice. In agreement, by in vivo quantitative imaging, we observed that treatment increased the number of LysM-EGFP cells traveling in tumor blood vessels and doubled the densities of both infiltrated LysM-EGFP monocytes and double-labeled EGFP/EYFP moDC. The treatment also led to an increased density of conventional cDCs2 subset together with a decrease of cDCs1 subset, necessary for the development of anti-tumor immunity. Finally, we describe differential spatial cell distributions and two immune cell-traveling routes into the brain. LysM-EGFP cells distributed as a gradient from the meninges towards the tumor whereas CD11c-EYFP/MHCII+ cells were located in the basal area of the tumor. Brain clearing also revealed a flow of CD11c-EYFP cells following the corpus callosum. CONCLUSION: We uncovered new features in the dynamics of innate immune cells in GBM-bearing mice and deciphered precisely the key populations, i.e., DC subsets controlling immune responses, that are affected by VEGF blockade. Since despite differences, human pathogenesis presents similarities with our mouse model, the data provide new insights into the effect of bevacizumab at the cellular level.

Thrombomodulin (TM) in tumor cell differentiation and periphery blood immune microenvironment in oral squamous cell carcinoma.

Thrombomodulin (TM, also known as CD141), which functions as an anticoagulant, is widely expressed on cell surface of a variety of cell types, including human blood cells as well as certain immune cells. To determine whether TM could be a potential marker for OSCC diagnosis as well as a molecular target for OSCC therapy, we examined the expression of TM in an oral cancer tissue microarray with 153 oral cancer tissues. Further, we also analyzed the expression of TM on DCs of 36 OSCC patients and 36 healthy donors. The expression of TM was determined using standard immunohistochemistry on a tissue microarray of 153 OSCC patients. Flow cytometric analyses were performed to determine the proportions of CD141+ DCs in the PBMC of 36 OSCC patients and 36 healthy donors. Clinicopathological correlations were performed based on the available clinical data. Our results showed that in the univariate analysis, high TM expression was significantly associated with well differentiation of tumor cells (P=.001), but not correlated with overall survival and disease-free survival (P>.05). In addition, CD141+ DCs were both present in OSCC patients and healthy donors with about 0.04%. There was no significant difference with the percentages of CD141+ DCs in the PBMC of OSCC patients and that of the normal control group (P>.05). This study indicates that TM expression might play the most critical role in the differentiation of OSCC tumors. Functional distinctions of CD141+ DCs in OSCC patients deserve further investigation to provide important therapeutic understandings for future immunotherapy.

Antigen cross-presentation by dendritic cell subsets: one general or all sergeants?

Antigen cross-presentation describes the process through which dendritic cells (DCs) acquire exogenous antigens for presentation on MHC class I molecules. The ability to cross-present has been thought of as a feature of specialized DC subsets. Emerging data, however, suggest that the cross-presenting ability of each DC subset is tuned by and dependent on several factors, such as DC location and activation status, and the type of antigen and inflammatory signals. Thus, we argue that capacity of cross-presentation is not an exclusive trait of one or several distinct DC subtypes, but rather a common feature of the DC family in both mice and humans. Understanding DC subset activation and antigen-presentation pathways might yield improved tools and targets to exploit the unique cross-presenting capacity of DCs in immunotherapy.

Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization.

Plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation.

DCs express receptors sensing microbial, danger or cytokine signals, which when triggered in combination drive DC maturation and functional polarization. Maturation was proposed to result from a discrete number of modifications in conventional DCs (cDCs), in contrast to a cell-fate conversion in plasmacytoid DCs (pDCs). cDC maturation is generally assessed by measuring cytokine production and membrane expression of MHC class II and co-stimulation molecules. pDC maturation complexity was demonstrated by functional genomics. Here, pDCs and cDCs were shown to undergo profound and convergent changes in their gene expression programs in vivo during viral infection. This observation was generalized to other stimulation conditions and DC subsets, by public microarray data analyses, PCR confirmation of selected gene expression profiles, and gene regulatory sequence bioinformatics analyses. Thus, maturation is a complex process similarly reshaping all DC subsets, including through the induction of a core set of NF-κB- or IFN-stimulated genes irrespective of stimuli.

Aligning bona fide dendritic cell populations across species.

Dendritic cells (DC) are professional antigen sensing and presenting cells that link innate and adaptive immunity. Consisting of functionally specialized subsets, they form a complex cellular network capable of integrating multiple environmental signals leading to immunity or tolerance. Much of DC research so far has been carried out in mice and increasing efforts are now being devoted to translating the findings into humans and other species. Recent studies have aligned these cellular networks across species at multiple levels from phenotype, gene expression program, ontogeny and functional specializations. In this review, we focus on recent advances in the definition of bona fide DC subsets across species. The understanding of functional similarities and differences of specific DC subsets in different animals not only brings light in the field of DC biology, but also paves the way for the design of future effective therapeutic strategies targeting these cells.

Quantitative trait loci mapping provides insights into the genetic regulation of dendritic cell numbers in mouse tissues.

To explore the influence of genetics on homeostatic regulation of dendritic cell (DC) numbers, we present a screen of DCs and their progenitors in lymphoid and non-lymphoid tissues in Collaborative Cross (CC) and Diversity Outbred (DO) mice. We report 30 and 71 loci with logarithm of the odds (LOD) scores >8.18 and ranging from 6.67 to 8.19, respectively. The analysis reveals the highly polygenic and pleiotropic architecture of this complex trait, including many of the previously identified genetic regulators of DC development and maturation. Two SNPs in genes potentially underlying variation in DC homeostasis, a splice variant in Gramd4 (rs235532740) and a missense variant in Orai3 (rs216659754), are confirmed by gene editing using CRISPR-Cas9. Gramd4 is a central regulator of DC homeostasis that impacts the entire DC lineage, and Orai3 regulates cDC2 numbers in tissues. Overall, the data reveal a large number of candidate genes regulating DC homeostasis in vivo.

Human Dendritic Cell Subset Isolation by Magnetic Bead Sorting: A Protocol to Efficiently Obtain Pure Populations.

Dendritic cells have been investigated for cell-based immunotherapy for various applications. The low abundance of dendritic cells in blood hampers their clinical application, resulting in the use of monocyte-derived dendritic cells as an alternative cell type. Limited knowledge is available regarding blood-circulating human dendritic cells, which can be divided into three subsets: type 2 conventional dendritic cells, type 1 conventional dendritic cells, and plasmacytoid dendritic cells. These subsets exhibit unique and desirable features for dendritic cell-based therapies. To enable efficient and reliable human research on dendritic cell subsets, we developed an efficient isolation protocol for the three human dendritic cell subsets, resulting in pure populations. The sequential steps include peripheral blood mononuclear cell isolation, magnetic-microbead lineage depletion (CD14, CD56, CD3, and CD19), and individual magnetic-microbead isolation of the three human dendritic cell subsets.

Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.

Catching Our Breath: Updates on the Role of Dendritic Cell Subsets in Asthma.

Dendritic cells (DCs), as potent antigen presenting cells, are known to play a central role in the pathophysiology of asthma. The understanding of DC biology has evolved over the years to include multiple subsets of DCs with distinct functions in the initiation and maintenance of asthma. Furthermore, asthma is increasingly recognized as a heterogeneous disease with potentially diverse underlying mechanisms. The goal of this review is to summarize the role of DCs and the various subsets therein in the pathophysiology of asthma and highlight some of the crucial animal models shaping the field today. Potential future avenues of investigation to address existing gaps in knowledge are discussed.

Research Progress of Dendritic Cell Surface Receptors and Targeting.

Dendritic cells are the only antigen-presenting cells capable of activating naive T cells in humans and mammals and are the most effective antigen-presenting cells. With deepening research, it has been found that dendritic cells have many subsets, and the surface receptors of each subset are different. Specific receptors targeting different subsets of DCs will cause different immune responses. At present, DC-targeted research plays an important role in the treatment and prevention of dozens of related diseases in the clinic. This article focuses on the current status of DC surface receptors and targeted applications.

Dendritic cell subsets in cancer immunity and tumor antigen sensing.

Dendritic cells (DCs) exhibit a specialized antigen-presenting function and play crucial roles in both innate and adaptive immune responses. Due to their ability to cross-present tumor cell-associated antigens to naïve T cells, DCs are instrumental in the generation of specific T-cell-mediated antitumor effector responses in the control of tumor growth and tumor cell dissemination. Within an immunosuppressive tumor microenvironment, DC antitumor functions can, however, be severely impaired. In this review, we focus on the mechanisms of DC capture and activation by tumor cell antigens and the role of the tumor microenvironment in shaping DC functions, taking advantage of recent studies showing the phenotype acquisition, transcriptional state and functional programs revealed by scRNA-seq analysis. The therapeutic potential of DC-mediated tumor antigen sensing in priming antitumor immunity is also discussed.

Protective interaction of human phagocytic APC subsets with Cryptococcus neoformans induces genes associated with metabolism and antigen presentation.

Cryptococcal meningitis is the most common cause of meningitis among HIV/AIDS patients in sub-Saharan Africa, and worldwide causes over 223,000 cases leading to more than 181,000 annual deaths. Usually, the fungus gets inhaled into the lungs where the initial interactions occur with pulmonary phagocytes such as dendritic cells and macrophages. Following phagocytosis, the pathogen can be killed or can replicate intracellularly. Previous studies in mice showed that different subsets of these innate immune cells can either be antifungal or permissive for intracellular fungal growth. Our studies tested phagocytic antigen-presenting cell (APC) subsets from the human lung against C. neoformans. Human bronchoalveolar lavage was processed for phagocytic APCs and incubated with C. neoformans for two hours to analyze the initial interactions and fate of the fungus, living or killed. Results showed all subsets (3 macrophage and 3 dendritic cell subsets) interacted with the fungus, and both living and killed morphologies were discernable within the subsets using imaging flow cytometry. Single cell RNA-seq identified several different clusters of cells which more closely related to interactions with C. neoformans and its protective capacity against the pathogen rather than discrete cellular subsets. Differential gene expression analyses identified several changes in the innate immune cell's transcriptome as it kills the fungus including increases of TNF-α (TNF) and the switch to using fatty acid metabolism by upregulation of the gene FABP4. Also, increases of TNF-α correlated to cryptococcal interactions and uptake. Together, these analyses implicated signaling networks that regulate expression of many different genes - both metabolic and immune - as certain clusters of cells mount a protective response and kill the pathogen. Future studies will examine these genes and networks to understand the exact mechanism(s) these phagocytic APC subsets use to kill C. neoformans in order to develop immunotherapeutic strategies to combat this deadly disease.

Whole Blood Dendritic Cell Cytokine Production Assay.

This protocol outlines a method for the timely detection of intracellular cytokines produced by activated dendritic cells (DC) in human whole blood. The quantification of cytokines is used to measure DC immune responsiveness, providing information on the breadth, strength, and DC subtypes responding spontaneously and to specific stimulation with toll-like receptor (TLR) ligands or parasite-infected erythrocytes. DC subsets, plasmacytoid DC, CD1c+ DC, CD141+ DC, and CD16+ DC, are examined in their natural environment of plasma and blood cells (erythrocytes, neutrophils, platelets, and leukocytes) enabling disease, medication, nutritional, and hematological effects on DC function to be examined in vaccine studies, ageing, health, and disease.