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Plasmacytoid dendritic cells (pDCs) are the main source of type I interferon (IFN-I) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DCs (tDCs), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. In the present report, we present a reporter mouse, pDC-Tom, designed through intersectional genetics based on unique Siglech and Pacsin1 coexpression in pDCs. The pDC-Tom mice specifically tagged pDCs and, on breeding with Zbtb46GFP mice, enabled transcriptomic profiling of all splenic DC types, unraveling diverging activation of pDC-like cells versus tDCs during a viral infection. The pDC-Tom mice also revealed initially similar but later divergent microanatomical relocation of splenic IFN+ versus IFN− pDCs during infection. The mouse models and specific gene modules we report here will be useful to delineate the physiological functions of pDCs versus other DC types.

While tuberculosis (TB) is a risk factor in HIV-1-infected individuals, the mechanisms by which Mycobacterium tuberculosis (Mtb), the agent of TB in humans, worsens HIV-1 pathogenesis still need to be fully elucidated. Recently, we showed that HIV-1 infection and spread are exacerbated in macrophages exposed to TB-associated microenvironments. Transcriptomic analysis of macrophages conditioned with medium of Mtb-infected human macrophages (cmMTB) revealed an up-regulation of the typeI interferon (IFN-I) pathway, characterized by the overexpression of IFN-inducible genes. Historically, IFN-I are well known for their antiviral functions, but our previous work showed that this is not the case in the context of coinfection with HIV-1. Here, we show that the IFN-I response signature in cmMTB-treated macrophages matches the one observed in the blood of active TB patients, and depends on the timing of incubation with cmMTB. This suggests that the timing of macrophage's exposure to IFN-I can impact their capacity to control HIV-1 infection. Strikingly, we found that cmMTB-treated macrophages are hyporesponsive to extrastimulation with exogenous IFN-I, used to mimic HIV-1 infection. Yet, depleting STAT1 by gene silencing to block the IFN-I signaling pathway reduced TB-induced exacerbation of HIV-1 infection. Altogether, by aiming to understand why TB-derived IFN-I preexposure of macrophages did not induce antiviral immunity against HIV-1, we demonstrated that these cells are hyporesponsive to exogenous IFN-I, a phenomenon that prevents macrophage activation against HIV-1.Mycobacterium tuberculosis induces hyporesponsiveness of the IFN-I signaling pathway in macrophages, leading to the exacerbation of HIV-1 replication.

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. While a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal physiological consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild type kidney. Transmission electron microscopy (TEM) of Tshz3+/lacZ glomeruli revealed ultrastructural defects. Compared to wild type, Tshz3+/lacZ mice showed no difference in their urine parameters but lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild type (WT; control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. Although a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney as well as in the proximal nephron tubules in neonatal mice. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild-type kidney. Transmission electron microscopy of Tshz3+/lacZ glomeruli revealed a reduced thickness of the glomerular basement membrane and a larger foot process width. Compared to wild type, Tshz3+/lacZ mice showed lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild-type (control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

International audience; Conventional type 1 dendritic cells (cDC1s) are critical for antitumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8 + T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the antitumor functions of cDC1s in immunogenic tumors are poorly understood. Using single-cell transcriptomics to examine the molecular pathways regulating intratumoral cDC1 maturation, we found nuclear factor κB (NF-κB) and interferon (IFN) pathways to be highly enriched in a subset of functionally mature cDC1s. We identified an NF-κB–dependent and IFN-γ–regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. By mapping the trajectory of intratumoral cDC1 maturation, we demonstrated the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IFN regulatory factor 1 (IRF1) in cDC1s, resulting in impaired expression of IFN-γ–responsive genes and consequently a failure to efficiently recruit and activate antitumoral CD8 + T cells. Last, we demonstrate the relevance of these findings to patients with melanoma, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development of new diagnostic and therapeutic approaches to improve cancer immunotherapy.

Conventional type 1 dendritic cells (cDC1s) are critical for anti-tumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8+ T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the anti-tumor functions of cDC1s are poorly understood. We mapped the molecular pathways regulating intra-tumoral cDC1 maturation using single cell RNA sequencing. We identified NF-κB and IFN pathways as being highly enriched in a subset of functionally mature cDC1s. The specific targeting of NF-κB or IFN pathways in cDC1s prevented the recruitment and activation of CD8+ T cells and the control of tumor growth. We identified an NF-κB-dependent IFNγ-regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. We used single cell transcriptomes to map the trajectory of intra-tumoral cDC1 maturation which revealed the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IRF1 in cDC1s, resulting in impaired expression of IFN-γ-responsive genes and consequently a failure to efficiently recruit and activate anti-tumoral CD8+ T cells. Finally, we demonstrate the relevance of these findings to cancer patients, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development new diagnostic and therapeutic approaches to improve cancer immunotherapy.One Sentence SummaryNF-κB and IRF1 coordinate intra-tumoral cDC1 maturation and control of immunogenic tumor growth.

International audience; Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.

Macrophages possess intrinsic tumoricidal activity, yet tumor-associated macrophages (TAMs) rapidly adopt an alternative phenotype within the tumor microenvironment that is marked by tumor-promoting immunosuppressive and trophic functions. The mechanisms that promote such TAM polarization remain poorly understood, but once identified, they may represent important therapeutic targets to block the tumor-promoting functions of TAMs and restore their anti-tumor potential. Here, we have characterized TAMs in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4-mediated reprogramming, including inhibition of IFNγ-induced gene expression. Genetic deletion of ABC transporters, which mediate cholesterol efflux, reverts the tumor-promoting functions of TAMs and reduces tumor progression. These studies reveal an unexpected role for membrane-cholesterol efflux in driving TAM-mediated tumor progression while pointing to a potentially novel anti-tumor therapeutic strategy. Goossens, Rodriguez-Vita et al. show that cancer cells scavenge membrane cholesterol from tumor-associated macrophages, resulting in their reprogramming toward an immune-suppressive and tumor-promoting phenotype. Targeting cholesterol efflux in macrophages counters this reprogramming and reduces tumor progression in a model of ovarian cancer.

Dendritic cells (DCs) play a key role in the orchestration of antitumor immune responses. The cDC1 subset was demonstrated as essential for tumor control and response to immunotherapies in mice, but its precise role in human is largely unexplored. In our study, we combined the analysis of large cohorts of fresh breast and ovarian primary tumors and of public transcriptomic data sets to elucidate human cDC1 functions and clinical impact compared with other DC subsets. We identified a key role for intratumoral cDC1 in producing selectively type III interferon (IFN-III), strongly associated with cytokines and chemokines promoting cytotoxic immune cell recruitment and activation. Of utmost importance, we also revealed a positive impact of IFN-III and cDC1 on patient outcome in many human cancers and identified TLR3-triggering as a therapeutic strategy to trigger IFN-III production by tumor-associated cDC1. These data may pave the way for new IFN or cDC1-targeting antitumor therapies. Citation Format: Margaux Hubert, Coline Couillault, Thien Phong Vu Manh, Vincent Ollion, Helene Lopez-Mestre, Nabil Rahmouni, Janice KIielbassa, Celine Rodriguez, Christophe Sajous, Benoit Dumont, Anne-Claire Doffin, Isabelle Treilleux, Olivier Tredan, Marc Dalod, Nathalie BendrissS-Vermare, Christophe Caux, Jenny Valladeau-Guilemomd. IFN-III is selectively produced by cDC1 and predicts good clinical outcome in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A70.

International audience; Dendritic cells play a key role in the orchestration of antitumor immune responses. The cDC1 (conventional dendritic cell 1) subset has been shown to be essential for antitumor responses and response to immunotherapy, but its precise role in humans is largely unexplored. Using a multidisciplinary approach, we demonstrate that human cDC1 play an important role in the antitumor immune response through their capacity to produce type III interferon (IFN-λ). By analyzing a large cohort of breast primary tumors and public transcriptomic datasets, we observed specific production of IFN-λ1 by cDC1. In addition, both IFN-λ1 and its receptor were associated with favorable patient outcomes. We show that IFN-III promotes a TH1 microenvironment through increased production of IL-12p70, IFN-γ, and cytotoxic lymphocyte–recruiting chemokines. Last, we showed that engagement of TLR3 is a therapeutic strategy to induce IFN-III production by tumor-associated cDC1. These data provide insight into potential IFN- or cDC1-targeting antitumor therapies.

Tumor-associated macrophages (TAM) have been shown to have important roles in the malignant progression of various cancers. However, macrophages also posses intrinsic tumoricidal activity and can promote the activity of cytotoxic lymphocytes, but they rapidly adopt an alternative phenotype within tumors, associated with immune-suppression and trophic functions that support tumor growth. The mechanisms that promote TAM polarization in the tumor-microenvironment remain poorly understood, these mechanisms may represent important therapeutic targets to block the tumor-promoting functions of TAM and restore their anti-tumor potential. Here we have characterized TAM in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and the depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4 mediated reprogramming while inhibiting IFNγ-induced gene expression. These studies reveal an unexpected role for tumor-induced membrane-cholesterol efflux in driving the IL-4 signaling and the tumor-promoting functions of TAM, while rendering them refractory to pro-inflammatory stimuli. Thus, preventing cholesterol efflux in TAM could represent a novel therapeutic strategy to block pro-tumor functions and restore anti-tumor immunity.

Endoplasmic reticulum (ER) stress triggers or amplifies inflammatory signals and cytokine production in immune cells. Upon the resolution of ER stress, the inducible phosphatase 1 cofactor GADD34 promotes the dephosphorylation of the initiation factor eIF2α, thereby enabling protein translation to resume. Several aminoguanidine compounds, such as guanabenz, perturb the eIF2α phosphorylation-dephosphorylation cycle and protect different cell or tissue types from protein misfolding and degeneration. We investigated how pharmacological interference with the eIF2α pathway could be beneficial to treat autoinflammatory diseases dependent on proinflammatory cytokines and type I interferons (IFNs), the production of which is regulated by GADD34 in dendritic cells (DCs). In mouse and human DCs and B cells, guanabenz prevented the activation of Toll-like receptor 9 (TLR9) by CpG oligodeoxynucleotides or DNA-immunoglobulin complexes in endosomes. In vivo, guanabenz protected mice from CpG oligonucleotide-dependent cytokine shock and decreased autoimmune symptom severity in a chemically induced model of systemic lupus erythematosus. However, we found that guanabenz exerted its inhibitory effect independently of GADD34 activity on eIF2α and instead decreased the abundance of CH25H, a cholesterol hydroxylase linked to antiviral immunity. Our results therefore suggest that guanabenz and similar compounds could be used to treat type I IFN-dependent pathologies and that CH25H could be a therapeutic target to control these diseases. published

Summary The ability to generate large numbers of distinct types of human dendritic cells (DCs) in vitro is critical for accelerating our understanding of DC biology and harnessing them clinically. We developed a DC differentiation method from human CD34+ precursors leading to high yields of plasmacytoid DCs (pDCs) and both types of conventional DCs (cDC1s and cDC2s). The identity of the cells generated in vitro and their strong homology to their blood counterparts were demonstrated by phenotypic, functional, and single-cell RNA-sequencing analyses. This culture system revealed a critical role of Notch signaling and GM-CSF for promoting cDC1 generation. Moreover, we discovered a pre-terminal differentiation state for each DC type, characterized by high expression of cell-cycle genes and lack of XCR1 in the case of cDC1. Our culture system will greatly facilitate the simultaneous and comprehensive study of primary, otherwise rare human DC types, including their mutual interactions., Graphical Abstract, Highlights • A CD34+ cell culture protocol yields large numbers of human pDCs and cDC1/2s • Notch signaling is critical for cDC1 generation and GM-CSF has a synergistic effect • scRNAseq confirms homology of in-vitro-derived DC types to their blood counterparts • CLEC9A-positive XCR1-negative cells were identified as immediate precursors of cDC1s, Balan et al. report a protocol to simultaneously generate large numbers of human pDCs, cDC1s, and cDC2s from cord blood and non-mobilized CD34+ progenitors. This culture system will enable experimental testing of mechanisms controlling the differentiation or functions of human DC types and their translational application to treat cancer.

Cytotoxic CD4 (CD4CTX) T cells are emerging as an important component of anti-viral and anti-tumor immunity but the molecular basis of their development remains poorly understood. In the context of human cytomegalovirus infection, a significant proportion of CD4 T cells displays cytotoxic functions. We observed that the transcriptional program of these cells was enriched in CD8 T cell lineage genes despite the absence of ThPOK downregulation. We further show that establishment of CD4CTX-specific transcriptional and epigenetic programs occurred in a stepwise fashion along the Th1-differentiation pathway. In vitro, prolonged activation of naive CD4 T cells in presence of Th1 polarizing cytokines led to the acquisition of perforin-dependent cytotoxic activity. This process was dependent on the Th1 transcription factor Runx3 and was limited by the sustained expression of ThPOK. This work elucidates the molecular program of human CD4CTXT cells and identifies potential targets for immunotherapy against viral infections and cancer., SCOPUS: ar.j, info:eu-repo/semantics/published

Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)–mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture–release–recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN‐I) during viral infections, in response to triggering of endosomal Toll‐like receptors (TLRs) 7 or 9 by viral single‐stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN‐I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN‐I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN‐I downstream of the TLR9‐to‐MyD88‐to‐IRF7 signaling pathway without requiring IFN‐I positive feedback, high IRF7 expression, or AP3‐driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN‐I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.

International audience; In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2a phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti-viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN-b mRNA transcription , while GADD34-dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA-activated cells.

International audience; Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.

Dendritic cells (DCs) are immune sentinels of the body and play a key role in the orchestration of the communication between the innate and the adaptive immune systems. DCs can polarize innate and adaptive immunity toward a variety of functions, sometimes with opposite roles in the overall control of immune responses (e.g., tolerance or immunosuppression versus immunity) or in the balance between various defense mechanisms promoting the control of different types of pathogens (e.g., antiviral versus antibacterial versus anti-worm immunity). These multiple DC functions result both from the plasticity of individual DC to exert different activities and from the existence of various DC subsets specialized in distinct functions. Functional genomics represents a powerful, unbiased, approach to better characterize these two levels of DC plasticity and to decipher its molecular regulation. Indeed, more and more experimental immunologists are generating high-throughput data in order to better characterize different states of DC based, for example, on their belonging to a specific subpopulation and/or on their exposure to specific stimuli and/or on their ability to exert a specific function. However, the interpretation of this wealth of data is severely hampered by the bottleneck of their bioinformatics analysis. Indeed, most experimental immunologists lack advanced computational or bioinformatics expertise and do not know how to translate raw gene expression data into potential biological meaning. Moreover, subcontracting such analyses is generally disappointing or financially not sustainable, since companies generally propose canonical analysis pipelines that are often unadapted for the structure of the data to analyze or for the precise type of questions asked. Hence, there is an important need of democratization of the bioinformatics analyses of gene expression profiling studies, in order to accelerate interpretation of the results by the researchers at the origin of the research project, of the data and who know best the underlying biology. This chapter will focus on the analysis of DC subset transcriptomes as measured by microarrays. We will show that simple bioinformatics procedures, applied one after the other in the framework of a pipeline, can lead to the characterization of DC subsets. We will develop two tutorials based on the reanalysis of public gene expression data. The first tutorial aims at illustrating a strategy for establishing the identity of DC subsets studied in a novel context, here their in vitro generation in cultures of human CD34(+) hematopoietic progenitors. The second tutorial aims at illustrating how to perform a posteriori bioinformatics analyses in order to evaluate the risk of contamination or of improper identification of DC subsets during preparation of biological samples, such that this information is taken into account in the final interpretation of the data and can eventually help to redesign the sampling strategy.

Dendritic cells (DCs) are immune sentinels of the body and play a key role in the orchestration of the communication between the innate and the adaptive immune systems. DCs can polarize innate and adaptive immunity toward a variety of functions, sometimes with opposite roles in the overall control of immune responses (e.g., tolerance or immunosuppression versus immunity) or in the balance between various defense mechanisms promoting the control of different types of pathogens (e.g., antiviral versus antibacterial versus anti-worm immunity). These multiple DC functions result both from the plasticity of individual DC to exert different activities and from the existence of various DC subsets specialized in distinct functions. Functional genomics represents a powerful, unbiased, approach to better characterize these two levels of DC plasticity and to decipher its molecular regulation. Indeed, more and more experimental immunologists are generating high-throughput data in order to better characterize different states of DC based, for example, on their belonging to a specific subpopulation and/or on their exposure to specific stimuli and/or on their ability to exert a specific function. However, the interpretation of this wealth of data is severely hampered by the bottleneck of their bioinformatics analysis. Indeed, most experimental immunologists lack advanced computational or bioinformatics expertise and do not know how to translate raw gene expression data into potential biological meaning. Moreover, subcontracting such analyses is generally disappointing or financially not sustainable, since companies generally propose canonical analysis pipelines that are often unadapted for the structure of the data to analyze or for the precise type of questions asked. Hence, there is an important need of democratization of the bioinformatics analyses of gene expression profiling studies, in order to accelerate interpretation of the results by the researchers at the origin of the research project, of the data and who know best the underlying biology. This chapter will focus on the analysis of DC subset transcriptomes as measured by microarrays. We will show that simple bioinformatics procedures, applied one after the other in the framework of a pipeline, can lead to the characterization of DC subsets. We will develop two tutorials based on the reanalysis of public gene expression data. The first tutorial aims at illustrating a strategy for establishing the identity of DC subsets studied in a novel context, here their in vitro generation in cultures of human CD34(+) hematopoietic progenitors. The second tutorial aims at illustrating how to perform a posteriori bioinformatics analyses in order to evaluate the risk of contamination or of improper identification of DC subsets during preparation of biological samples, such that this information is taken into account in the final interpretation of the data and can eventually help to redesign the sampling strategy.

SummaryType I interferons (IFNs) are central to antiviral defense, but how they orchestrate immune cell function is incompletely understood. We determined that IFNs produced during murine cytomegalovirus (MCMV) infection differentially affect dendritic cells (DCs) and natural killer (NK) cells. IFNs induce cell-intrinsic responses in DCs, activating antiproliferative, antiviral, and lymphocyte-activating gene networks, consistent with high activity of the transcription factor STAT1 in these cells. By comparison, NK cells exhibit lower STAT1 expression and reduced IFN responsiveness. Rather, IFNs indirectly affect NK cells by inducing IL-15, which activates the transcription factor E2F and stimulates genes promoting cell expansion. IFN cell-intrinsic responses are necessary in DCs, but not NK cells, for MCMV resistance. Thus, sensitivity to IFN-induced cytokines and differences in IFN receptor signaling program immune cells to mount distinct responses that promote viral control.

Subsets of dendritic cells (DCs) have been described according to their functions and anatomical locations. Conventional DC subsets are defined by reciprocal expression of CD11b and CD8α in lymphoid tissues (LT), and of CD11b and CD103 in non-LT (NLT). Spleen CD8α+ and dermal CD103+ DCs share a high efficiency for Ag cross-presentation and a developmental dependency on specific transcription factors. However, it is not known whether all NLT-derived CD103+ DCs and LT-resident CD8α+ DCs are similar despite their different anatomical locations. XCR1 was previously described as exclusively expressed on mouse spleen CD8α+ DCs and human blood BDCA3+ DCs. In this article, we showed that LT-resident CD8α+ DCs and NLT-derived CD103+ DCs specifically express XCR1 and are characterized by a unique transcriptional fingerprint, irrespective of their tissue of origin. Therefore, CD8α+ DCs and CD103+ DCs belong to a common DC subset which is unequivocally identified by XCR1 expression throughout the body.

The gut is a major barrier against microbes and encloses various innate lymphoid cells (ILCs), including two subsets expressing the natural cytotoxicity receptor NKp46. A subset of NKp46(+) cells expresses retinoic acid receptor-related orphan receptor γt (RORγt) and produces IL-22, like lymphoid tissue inducer (LTi) cells. Other NKp46(+) cells lack RORγt and produce IFN-γ, like conventional Natural Killer (cNK) cells. The identity, the regulation and the in vivo functions of gut NKp46(+) ILCs largely remain to be unravelled. Using pan-genomic profiling, we showed here that small intestine (SI) NKp46(+)RORγt(-) ILCs correspond to SI NK cells. Conversely, we identified a transcriptional programme conserved in fetal LTi cells and adult SI NKp46(+)RORγt(+) and NKp46(-)RORγt(+) ILCs. We also demonstrated that the IL-1β/IL-1R1/MyD88 pathway, but not the commensal flora, drove IL-22 production by NKp46(+)RORγt(+) ILCs. Finally, oral Listeria monocytogenes infection induced IFN-γ production in SI NK and IL-22 production in NKp46(+)RORγt(+) ILCs, but only IFN-γ contributed to control bacteria dissemination. NKp46(+) ILC heterogeneity is thus associated with subset-specific transcriptional programmes and effector functions that govern their implication in gut innate immunity.

SummaryHere we describe a lineage reprogramming system consisting of a B cell line with an estradiol-inducible form of C/EBPα where cells can be converted into macrophage-like cells at 100% efficiency within 2 to 3 days. The reprogrammed cells are larger, contain altered organelle and cytoskeletal structures, are phagocytic, and exhibit an inflammatory response. Time-lapse experiments showed that the cells acquire a macrophage morphology and increased migratory activity as early as 10 hr. During induction, thousands of genes become up- or downregulated, including several dozen transcription and chromatin-remodeling factors. Time-limited exposure of cells to the inducer showed that the reprogrammed cells become transgene independent within 1 to 2 days. The reprogramming can be inhibited, at least partially, by perturbation experiments with B cell and macrophage transcription factors. The tightness, robustness, and speed of the system described make it a versatile tool to study biochemical and biological aspects of lineage reprogramming.

Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1+ DCs, and between mouse and human Langerhans cells., Highlights • Rigorous phenotypic identification of human CD141highXCR1+ dermal DCs is challenging. • Comparative genomics enables rigorous identification of cell populations. • Human dermal CD141highXCR1+ DCs are homologous to human blood and mouse XCR1+ DCs. • Human Langerhans cells are homologous to mouse Langerhans cells. • Langerhans cells selectively express DC rather than macrophage gene signatures.

Background Recent advances in the analysis of high-throughput expression data have led to the development of tools that scaled-up their focus from single-gene to gene set level. For example, the popular Gene Set Enrichment Analysis (GSEA) algorithm can detect moderate but coordinated expression changes of groups of presumably related genes between pairs of experimental conditions. This considerably improves extraction of information from high-throughput gene expression data. However, although many gene sets covering a large panel of biological fields are available in public databases, the ability to generate home-made gene sets relevant to one’s biological question is crucial but remains a substantial challenge to most biologists lacking statistic or bioinformatic expertise. This is all the more the case when attempting to define a gene set specific of one condition compared to many other ones. Thus, there is a crucial need for an easy-to-use software for generation of relevant home-made gene sets from complex datasets, their use in GSEA, and the correction of the results when applied to multiple comparisons of many experimental conditions. Result We developed BubbleGUM (GSEA Unlimited Map), a tool that allows to automatically extract molecular signatures from transcriptomic data and perform exhaustive GSEA with multiple testing correction. One original feature of BubbleGUM notably resides in its capacity to integrate and compare numerous GSEA results into an easy-to-grasp graphical representation. We applied our method to generate transcriptomic fingerprints for murine cell types and to assess their enrichments in human cell types. This analysis allowed us to confirm homologies between mouse and human immunocytes. Conclusions BubbleGUM is an open-source software that allows to automatically generate molecular signatures out of complex expression datasets and to assess directly their enrichment by GSEA on independent datasets. Enrichments are displayed in a graphical output that helps interpreting the results. This innovative methodology has recently been used to answer important questions in functional genomics, such as the degree of similarities between microarray datasets from different laboratories or with different experimental models or clinical cohorts. BubbleGUM is executable through an intuitive interface so that both bioinformaticians and biologists can use it. It is available at http://www.ciml.univ-mrs.fr/applications/BubbleGUM/index.html. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2012-4) contains supplementary material, which is available to authorized users.

Computational cost of BubbleMap. (DOCX 15 kb)

Mononuclear phagocytes are organized in a complex system of ontogenically and functionally-distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections.We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey, indicating conservation of the genetic support for mononuclear phagocyte organization throughout jawed vertebrates but likely not in agnathans. Altogether this work provides molecular clues to the definition and functions of mononuclear phagocyte subsets across vertebrates which shall be useful to rigorously identify these cells and to design universal strategies to manipulate them in many target species towards the goal to reach and maintain global health.

Interleukin-4 boosts the capacity of dendritic cells to present endogenous antigens on MHC II and to resist bacterial infection through a mechanism shown to be partially dependent on RUFY4 expression., Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.

It provides technical information about the methods used in BubbleGUM and is complementary with the online User Guide ( http://www.ciml.univ-mrs.fr/applications/BubbleGUM/index.html ) which is dedicated to provide practical description of the tool. (PDF 1362 kb)

International audience; The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1+ DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1+ human DC. Assessment of the immunoactivation potential of XCR1+ human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1+ and XCR1− human DC in CD34+ progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1− CD34-DC are similar to canonical MoDC, whereas XCR1+ CD34-DC resemble XCR1+ blood DC (bDC). XCR1+ DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1+ DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1+ CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1+ bDC. Hence, it is feasible to generate high numbers of bona fide XCR1+ human DC in vitro as a model to decipher the functions of XCR1+ bDC and as a potential source of XCR1+ DC for clinical use.

Liver is a major regulator of lipid metabolism and adaptation to fasting, a process involving PPARalpha activation. We recently showed that the Vnn1 gene is a PPARalpha target gene in liver and that release of the Vanin-1 pantetheinase in serum is a biomarker of PPARalpha activation. Here we set up a screen to identify new regulators of adaptation to fasting using the serum Vanin-1 as a marker of PPARalpha activation. Mutagenized mice were screened for low serum Vanin-1 expression. Functional interactions with PPARalpha were investigated by combining transcriptomic, biochemical and metabolic approaches. We characterized a new mutant mouse in which hepatic and serum expression of Vanin-1 is depressed. This mouse carries a mutation in the HMG domain of the Sox17 transcription factor. Mutant mice display a metabolic phenotype featuring lipid abnormalities and inefficient adaptation to fasting. Upon fasting, a fraction of the PPARα-driven transcriptional program is no longer induced and associated with impaired fatty acid oxidation. The transcriptional phenotype is partially observed in heterozygous Sox17+/- mice. In mutant mice, the fasting phenotype but not all transcriptomic signature is rescued by the administration of the PPARalpha agonist fenofibrate. These results identify a novel role for Sox17 in adult liver as a modulator of the metabolic adaptation to fasting.