SummaryDendritic 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.

Dendritic cells (DCs) and monocyte-derived macrophages (M s) are key components of intestinal immunity. However, the lack of surface markers differentiating M s from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, M s 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 M s and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly-6C hi monocytes to intestinal M s. 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 M s endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. Eur. J. Immunol. 2012. 42: 3150-3166 HIGHLIGHTS 3151 IntroductionThe intestinal lamina propria (LP) contains cells that express high levels of CX 3 CR1, the receptor for the fractalkine chemokine [1,2]. Based on their monocytic origin and on their inability to migrate to the mesenteric lymph nodes (MLNs) such CX 3 CR1 hi cells have been defined as macrophages (M s) [1][2][3]. CX 3 CR1 hi M s contribute to the intestinal LP homeostasis through the production of anti-inflammatory cytokines and the clearance of commensal bacteria that breach the epithelial barrier [4]. In contrast, during intestinal inflammation, microenvironmental signals promote the differentiation of extravasated monocytes into proinflammatory M s with the ability to produce interleukin (IL)-12, IL-23, tumor necrosis (TNF)-α and inducible nitric oxide synthase (iNOS) [5][6][7]. However, little is known about the developmental trajectories that lead extravasated monocytes to either antior proinflammatory intestinal M s. This is primarily due to the fact that a surface marker permitting unequivocal identification of M s within the intestine and their distinction from dendritic cells (DCs) is lacking. The interstitial DCs (Int-DCs) present throughout the LP derive from blood precursors known as pre-DCs [2]. Under steady-state conditions, the Int-DCs found in the intestinal LP induce oral tolerance by carrying antigens originating from food or from harmless bacteria to the MLNs [8,9]. The CD103 + Int-DCs found in the steady-state LP have the selective ability to express aldehyde dehydrogenase (ALDH) and thereby produce retinoic acid (RA). As a result, upon migration to MLNs they trigger the differentiation of naive CD4 + T cells specific for food and microbiota antigens into induced Foxp3 + regulatory T (iTreg) cells [10][11][12][13]. In contrast, the Int-DCs that develop in inflamed LP upon exposure to pathogens lose their capacity to generate iTreg cells and, upon migration to the MLNs, trigger the differentiation of naive, antigen-responsive CD4 + T cells into T helper type 1 (Th1) cells that are specific for the invading patho...

Highlights d Type I interferon drives differentiation of inf-cDC2s that closely resemble MCs d Inf-cDC2s prime CD4 + and CD8 + T cells, whereas MCs lack APC function d Inf-cDC2s internalize antibody-complexed antigen via Fc receptors d IRF8 controls maturation gene module in inf-cDC2s

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.

The identification of intestinal macrophages (mφs) and dendritic cells (DCs) is a matter of intense debate. Although CD103+ mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103− MPs remain controversial. We show here that intestinal CD103−CD11b+ MPs can be separated clearly into DCs and mφs based on phenotype, gene profile, and kinetics. CD64−CD103−CD11b+ MPs are classical DCs, being derived from Flt3 ligand-dependent, DC-committed precursors, not Ly6Chi monocytes. Surprisingly, a significant proportion of these CD103−CD11b+ DCs express CCR2 and there is a selective decrease in CD103−CD11b+ DCs in mice lacking this chemokine receptor. CCR2+CD103− DCs are present in both the murine and human intestine, drive interleukin (IL)-17a production by T cells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2+ DCs that is involved in priming mucosal T helper type 17 (Th17) responses.

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.

While central nervous system (CNS) microglia have been studied extensively, surprisingly little is known about macrophages populating the peripheral nervous system (PNS Macs).We performed ontogenic, transcriptomic and spatial characterization of sciatic nerve Macs (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 get replaced by bone marrow-derived Macs over time. Using single-cell profiling, we identified a tissue-specific core signature of snMacs and found two spatially-separated snMacs: Relmα + Mgl1 + snMacs in the epineurium and Relmα -Mgl1-snMacs in the endoneurium. Globally, snMacs lack most core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. Single-cell transcriptomics revealed that in response to injury both snMacs respond differently and that the PNS, in contrast to the CNS, is permissive to prolonged engraftment of monocytederived Macs recruited upon injury.

SummaryHeterogeneity 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.