Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.

The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.

The Transcription Factor ZEB2 Is Required to Maintain the Tissue-Specific Identities of Macrophages.

Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.

IRF8 Transcription Factor Controls Survival and Function of Terminally Differentiated Conventional and Plasmacytoid Dendritic Cells, Respectively.

Interferon regulatory factor-8 (IRF8) has been proposed to be essential for development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) and remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells are designated "terminal selectors." Using BM chimeras, conditional Irf8(fl/fl) mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene-expression profile leading to increased T cell stimulatory function but decreased type 1 interferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s, and pDCs.

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.

PeacoQC: Peak-based selection of high quality cytometry data.

In cytometry analysis, a large number of markers is measured for thousands or millions of cells, resulting in high-dimensional datasets. During the measurement of these samples, erroneous events can occur such as clogs, speed changes, slow uptake of the sample etc., which can influence the downstream analysis and can even lead to false discoveries. As these issues can be difficult to detect manually, an automated approach is recommended. In order to filter these erroneous events out, we created a novel quality control algorithm, Peak Extraction And Cleaning Oriented Quality Control (PeacoQC), that allows for automated cleaning of cytometry data. The algorithm will determine density peaks per channel on which it will remove low quality events based on their position in the isolation tree and on their mean absolute deviation distance to these density peaks. To evaluate PeacoQC's cleaning capability, it was compared to three other existing quality control algorithms (flowAI, flowClean and flowCut) on a wide variety of datasets. In comparison to the other algorithms, PeacoQC was able to filter out all different types of anomalies in flow, mass and spectral cytometry data, while the other methods struggled with at least one type. In the quantitative comparison, PeacoQC obtained the highest median balanced accuracy and a similar running time compared to the other algorithms while having a better scalability for large files. To ensure that the parameters chosen in the PeacoQC algorithm are robust, the cleaning tool was run on 16 public datasets. After inspection, only one sample was found where the parameters should be further optimized. The other 15 datasets were analyzed correctly indicating a robust parameter choice. Overall, we present a fast and accurate quality control algorithm that outperforms existing tools and ensures high-quality data that can be used for further downstream analysis. An R implementation is available.

Epicutaneous sensitization to house dust mite allergen requires interferon regulatory factor 4-dependent dermal dendritic cells.

BACKGROUND: Exposure to allergens, such as house dust mite (HDM), through the skin often precedes allergic inflammation in the lung. It was proposed that TH2 sensitization through the skin occurs when skin barrier function is disrupted by, for example, genetic predisposition, mechanical damage, or the enzymatic activity of allergens. OBJECTIVE: We sought to study how HDM applied to unmanipulated skin leads to TH2 sensitization and to study which antigen-presenting cells mediate this process. METHODS: HDM was applied epicutaneously by painting HDM on unmanipulated ear skin or under an occlusive tape. HDM challenge was through the nose. Mouse strains lacking different dendritic cell (DC) populations were used, and 1-DER T cells carrying a transgenic T-cell receptor reactive to Der p 1 allergen were used as a readout for antigen presentation. The TH2-inducing capacity of sorted skin-derived DC subsets was determined by means of adoptive transfer to naive mice. RESULTS: Epicutaneous HDM application led to TH2 sensitization and eosinophilic airway inflammation upon intranasal HDM challenge. Skin sensitization did not require prior skin damage or enzymatic activity within HDM extract, yet was facilitated by applying the allergen under an occlusive tape. Primary proliferation of 1-DER T cells occurred only in the regional skin-draining lymph nodes. Epicutaneous sensitization was found to be driven by 2 variants of interferon regulatory factor 4-dependent dermal type 2 conventional DC subsets and not by epidermal Langerhans cells. CONCLUSION: These findings identify skin type 2 conventional DCs as crucial players in TH2 sensitization to common inhaled allergens that enter the body through the skin and can provoke features of allergic asthma.

CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis.

Dendritic cells (DCs) and monocyte-derived macrophages (MΦs) are key components of intestinal immunity. However, the lack of surface markers differentiating MΦs from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, MΦs can be distinguished from DCs in the intestine of both mice and humans. On that basis, we revisit the phenotype of intestinal DCs in the absence of contaminating MΦs and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly-6C(hi) monocytes to intestinal MΦs. We determine how inflammation impacts this pathway and show that T cell-mediated colitis is associated with massive recruitment of monocytes to the intestine and the mesenteric lymph node (MLN). There, these monocytes differentiate into inflammatory MΦs endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. In the MLNs, inflammatory MΦs are located in the T-cell zone and trigger the induction of proinflammatory T cells. Finally, T cell-mediated colitis develops irrespective of intestinal DC migration, an unexpected finding supporting an important role for MLN-resident inflammatory MΦs in the etiology of T cell-mediated colitis.

Profiling peripheral nerve macrophages reveals two macrophage subsets with distinct localization, transcriptome and response to injury.

While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relmα+Mgl1+ snMacs in the epineurium and Relmα-Mgl1- snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.

Myocarditis Elicits Dendritic Cell and Monocyte Infiltration in the Heart and Self-Antigen Presentation by Conventional Type 2 Dendritic Cells.

Autoimmune myocarditis often leads to dilated cardiomyopathy (DCM). Although T cell reactivity to cardiac self-antigen is common in the disease, it is unknown which antigen presenting cell (APC) triggers autoimmunity. Experimental autoimmune myocarditis (EAM) was induced by immunizing mice with α-myosin loaded bone marrow APCs cultured in GM-CSF. APCs found in such cultures include conventional type 2 CD11b+ cDCs (GM-cDC2s) and monocyte-derived cells (GM-MCs). However, only α-myosin loaded GM-cDC2s could induce EAM. We also studied antigen presenting capacity of endogenous type 1 CD24+ cDCs (cDC1s), cDC2s, and MCs for α-myosin-specific TCR-transgenic TCR-M CD4+ T cells. After EAM induction, all cardiac APCs significantly increased and cDCs migrated to the heart-draining mediastinal lymph node (LN). Primarily cDC2s presented α-myosin to TCR-M cells and induced Th1/Th17 differentiation. Loss of IRF4 in Irf4fl/fl.Cd11cCre mice reduced MHCII expression on GM-cDC2s in vitro and cDC2 migration in vivo. However, partly defective cDC2 functions in Irf4fl/fl.Cd11cCre mice did not suppress EAM. MCs were the largest APC subset in the inflamed heart and produced pro-inflammatory cytokines. Targeting APC populations could be exploited in the design of new therapies for cardiac autoimmunity.

Myocardial Infarction Primes Autoreactive T Cells through Activation of Dendritic Cells.

Peripheral tolerance is crucial for avoiding activation of self-reactive T cells to tissue-restricted antigens. Sterile tissue injury can break peripheral tolerance, but it is unclear how autoreactive T cells get activated in response to self. An example of a sterile injury is myocardial infarction (MI). We hypothesized that tissue necrosis is an activator of dendritic cells (DCs), which control tolerance to self-antigens. DC subsets of a murine healthy heart consisted of IRF8-dependent conventional (c)DC1, IRF4-dependent cDC2, and monocyte-derived DCs. In steady state, cardiac self-antigen α-myosin was presented in the heart-draining mediastinal lymph node (mLN) by cDC1s, driving the proliferation of antigen-specific CD4+ TCR-M T cells and their differentiation into regulatory cells (Tregs). Following MI, all DC subsets infiltrated the heart, whereas only cDCs migrated to the mLN. Here, cDC2s induced TCR-M proliferation and differentiation into interleukin-(IL)-17/interferon-(IFN)γ-producing effector cells. Thus, cardiac-specific autoreactive T cells get activated by mature DCs following myocardial infarction.

The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity.

Various steady state and inflamed tissues have been shown to contain a heterogeneous DC population consisting of developmentally distinct subsets, including cDC1s, cDC2s and monocyte-derived DCs, displaying differential functional specializations. The identification of functionally distinct tumour-associated DC (TADC) subpopulations could prove essential for the understanding of basic TADC biology and for envisaging targeted immunotherapies. We demonstrate that multiple mouse tumours as well as human tumours harbour ontogenically discrete TADC subsets. Monocyte-derived TADCs are prominent in tumour antigen uptake, but lack strong T-cell stimulatory capacity due to NO-mediated immunosuppression. Pre-cDC-derived TADCs have lymph node migratory potential, whereby cDC1s efficiently activate CD8+ T cells and cDC2s induce Th17 cells. Mice vaccinated with cDC2s displayed a reduced tumour growth accompanied by a reprogramming of pro-tumoural TAMs and a reduction of MDSCs, while cDC1 vaccination strongly induces anti-tumour CTLs. Our data might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors.