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Developmental cues for the different dendritic cell (DC) subsets in the intestine are yet to be defined. Here the authors show that TGFβR1 signalling is needed for development of CD103+CD11b+ intestinal DCs from CD103−CD11b+ cells and that they contribute to the generation of Th17 and regulatory T cells

Organic-inorganic hybrid materials are attractive due to the combination of properties from the two distinct types of materials. In this work, transparent titania-polydimethylsiloxane hybrid materials with up to 15.5 vol. % TiO2 content were prepared by an in situ non-aqueous method using titanium (IV) isopropoxide and hydroxy-terminated polydimethylsiloxane as precursors. Spectroscopy (Fourier transform infrared, Raman, Ultraviolet-visible, ellipsometry) and small-angle X-ray scattering analysis allowed to describe in detail the structure and the optical properties of the nanocomposites. Titanium alkoxide was successfully used as a cross-linker and titania-like nanodomains with an average size of approximately 4 nm were shown to form during the process. The resulting hybrid nanocomposites exhibit high transparency and tunable refractive index from 1.42 up to 1.56, depending on the titania content.

A simple, robust and versatile hydrothermal synthesis route to in situ functionalized TiO2 nanoparticles was developed using titanium(IV) isopropoxide as Ti-precursor and selected silane coupling agents (3-aminopropyltriethoxysilane (APTES), 3-(2-aminoethylamino)propyldimethoxymethylsilane (AEAPS), and n-decyltriethoxysilane (DTES)). Spherical nanoparticles (ca. 9 nm) with narrow size distribution were obtained by using DTES or by synthesis performed without silane coupling agents. Rod-like nanoparticles along with 9 nm spherical nanoparticles were formed using aminosilane coupling agents because of a combination of oriented attachment of nanoparticles and specific adsorption of the aminosilane on crystallographic faces of anatase nanoparticles. The nanoparticles were functionalized in situ and became hydrophobic as silanes reacted to form covalent bonds on the surface of TiO2. The versatility of the aqueous synthesis route was demonstrated, and by selecting the type of silane coupling agent the surface properties of the TiO2 nanoparticles could be tailored. This synthesis route has been further developed into a two-step synthesis to TiO2–SiO2 core–shell nanoparticles. Combustion of the silane coupling agents up to 700 °C leads to the formation of a nanometric amorphous SiO2 layer, preventing growth and phase transition of the in situ functionalized nanoparticles.

Cross-presentation by MHCI is optimally efficient in type 1 dendritic cells (DC) due to their high capacity for antigen processing. However, through specific pathways, other DCs, such as type 2 DCs and inflammatory DCs (iDCs) can also cross-present antigens. FcγR-mediated uptake by type 2 DC and iDC subsets mediates antibody-dependent cross-presentation and activation of CD8 + T cell responses. Here, an important role for the p84 regulatory subunit of PI3Kγ in mediating efficient cross-presentation of exogenous antigens in otherwise inefficient cross-presenting cells, such as type 2 DCs and GM-CSF-derived iDCs is identified. FcγR-mediated cross-presentation is shown in type 2 and iDCs depend on the enzymatic activity of the p84/p110γ complex of PI3Kγ, which controls the activity of the NADPH oxidase NOX2 and ROS production in murine spleen type 2 DCs and GM-CSF-derived iDCs. In contrast, p84/p110γ is largely dispensable for cross-presentation by type 1 DCs. These findings suggest that PI3Kγ-targeted therapies, currently considered for oncological practice, may interfere with the ability of type 2 DCs and iDCs to cross-present antigens contained in immune complexes., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Type I and III interferons (IFNs) are essential for antiviral immunity and act through two different but complimentary pathways. First, IFNs activate intracellular antimicrobial programs by triggering the upregulation of a broad repertoire of viral restriction factors. Second, IFNs activate innate and adaptive immunity. Dysregulation of IFN production can lead to severe immune system dysfunction. It is thus crucial to identify and characterize the cellular sources of IFNs, their effects, and their regulation to promote their beneficial effects and limit their detrimental effects, which can depend on the nature of the infected or diseased tissues, as we will discuss. Plasmacytoid dendritic cells (pDCs) can produce large amounts of all IFN subtypes during viral infection. pDCs are resistant to infection by many different viruses, thus inhibiting the immune evasion mechanisms of viruses that target IFN production or their downstream responses. Therefore, pDCs are considered essential for the control of viral infections and the establishment of protective immunity. A thorough bibliographical survey showed that, in most viral infections, despite being major IFN producers, pDCs are actually dispensable for host resistance, which is achieved by multiple IFN sources depending on the tissue. Moreover, primary innate and adaptive antiviral immune responses are only transiently affected in the absence of pDCs. More surprisingly, pDCs and their IFNs can be detrimental in some viral infections or autoimmune diseases. This makes the conservation of pDCs during vertebrate evolution an enigma and thus raises outstanding questions about their role not only in viral infections but also in other diseases and under physiological conditions., (© 2024. The Author(s).)

Deciphering the initial steps of SARS-CoV-2 infection, that influence COVID-19 outcomes, is challenging because animal models do not always reproduce human biological processes and in vitro systems do not recapitulate the histoarchitecture and cellular composition of respiratory tissues. To address this, we developed an innovative ex vivo model of whole human lung infection with SARS-CoV-2, leveraging a lung transplantation technique. Through single-cell RNA-seq, we identified that alveolar and monocyte-derived macrophages (AMs and MoMacs) were initial targets of the virus. Exposure of isolated lung AMs, MoMacs, classical monocytes and non-classical monocytes (ncMos) to SARS-CoV-2 variants revealed that while all subsets responded, MoMacs produced higher levels of inflammatory cytokines than AMs, and ncMos contributed the least. A Wuhan lineage appeared to be more potent than a D614G virus, in a dose-dependent manner. Amidst the ambiguity in the literature regarding the initial SARS-CoV-2 cell target, our study reveals that AMs and MoMacs are dominant primary entry points for the virus, and suggests that their responses may conduct subsequent injury, depending on their abundance, the viral strain and dose. Interfering on virus interaction with lung macrophages should be considered in prophylactic strategies., (© 2024. The Author(s).)

Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage., (© 2024. The Author(s).)

Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet + cDC2As and T-bet - cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H + pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H - CD8α + pre-cDC2As in tissues, which differentiated into T-bet + cDC2As. In contrast, a Siglec-H - fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet - cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment., (© 2024. The Author(s).)

Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony-stimulating factor (M-CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M-CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon-γ. This depended upon M-CSF-induced myelopoiesis leading to IL15Rα-mediated presentation of IL-15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M-CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G-CSF-mobilized hematopoietic stem and progenitor cells. Together, M-CSF-induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M-CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host-directed therapies., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

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 non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34 + cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34 + cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. 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 co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

In response to the increasing demand for lung transplantation, ex vivo lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4 + and CD8 + T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gouin, Vu Manh, Jouneau, Bevilacqua, De Wolf, Glorion, Hannouche, Urien, Estephan, Roux, Magnan, Le Guen, Da Costa, Chevalier, Descamps, Schwartz-Cornil, Dalod and Sage.)

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 Zbtb46 GFP 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., (© 2023. The Author(s).)

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cells that recognize tumor antigens are crucial for mounting antitumor immune responses. Induction of antitumor T cells in immunogenic tumors depends on STING, the intracellular innate immune receptor for cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and related cyclic dinucleotides (CDNs). However, the optimal way to leverage STING activation in nonimmunogenic tumors is still unclear. Here, we show that cGAMP delivery by intratumoral injection of virus-like particles (cGAMP-VLP) led to differentiation of circulating tumor-specific T cells, decreased tumor regulatory T cells (T regs ), and antitumoral responses that synergized with PD1 blockade. By contrast, intratumoral injection of the synthetic CDN ADU-S100 led to tumor necrosis and systemic T cell activation but simultaneously depleted immune cells from injected tumors and induced minimal priming of circulating tumor-specific T cells. The antitumor effects of cGAMP-VLP required type 1 conventional dendritic cells (cDC1), whereas ADU-S100 eliminated cDC1 from injected tumors. cGAMP-VLP preferentially targeted STING in dendritic cells at a 1000-fold smaller dose than ADU-S100. Subcutaneous administration of cGAMP-VLP showed synergy when combined with PD1 blockade or a tumor T reg -depleting antibody to elicit systemic tumor-specific T cells and antitumor activity, leading to complete and durable tumor eradication in the case of tumor T reg depletion. These findings show that cell targeting of STING stimulation shapes the antitumor T cell response and identify a therapeutic strategy to enhance T cell-targeted immunotherapy.

Dendritic cells (DCs) represent one of the most important immune cell subsets in preventing the host from pathogen invasion by promoting both innate and adaptive immunity. Most research on human dendritic cells has focused on the easy-to-obtain dendritic cells derived in vitro from monocytes (MoDCs). However, many questions remain unanswered regarding the role of different dendritic cell types. The investigation of their roles in human immunity is hampered by their rarity and fragility, which especially holds true for type 1 conventional dendritic cells (cDC1s) and for plasmacytoid dendritic cells (pDCs). In vitro differentiation from hematopoietic progenitors emerged as a common way to produce different DC types, but the efficiency and reproducibility of these protocols needed to be improved and the extent to which the DCs generated in vitro resembled their in vivo counterparts required a more rigorous and global assessment. Here, we describe a cost-effective and robust in vitro differentiation system for the production of cDC1s and pDCs equivalent to their blood counterparts, from cord blood CD34 + hematopoietic stem cells (HSCs) cultured on a stromal feeder layer with a combination of cytokines and growth factors., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Dendritic cells (DCs) orchestrate innate and adaptive immunity, by translating the sensing of distinct danger signals into the induction of different effector lymphocyte responses, to induce the defense mechanisms the best suited to face the threat. Hence, DCs are very plastic, which results from two key characteristics. First, DCs encompass distinct cell types specialized in different functions. Second, each DC type can undergo different activation states, fine-tuning its functions depending on its tissue microenvironment and the pathophysiological context, by adapting the output signals it delivers to the input signals it receives. Hence, to better understand DC biology and harness it in the clinic, we must determine which combinations of DC types and activation states mediate which functions and how.To decipher the nature, functions, and regulation of DC types and their physiological activation states, one of the methods that can be harnessed most successfully is ex vivo single-cell RNA sequencing (scRNAseq). However, for new users of this approach, determining which analytics strategy and computational tools to choose can be quite challenging, considering the rapid evolution and broad burgeoning in the field. In addition, awareness must be raised on the need for specific, robust, and tractable strategies to annotate cells for cell type identity and activation states. It is also important to emphasize the necessity of examining whether similar cell activation trajectories are inferred by using different, complementary methods. In this chapter, we take these issues into account for providing a pipeline for scRNAseq analysis and illustrating it with a tutorial reanalyzing a public dataset of mononuclear phagocytes isolated from the lungs of naïve or tumor-bearing mice. We describe this pipeline step-by-step, including data quality controls, dimensionality reduction, cell clustering, cell cluster annotation, inference of the cell activation trajectories, and investigation of the underpinning molecular regulation. It is accompanied with a more complete tutorial on GitHub. We hope that this method will be helpful for both wet lab and bioinformatics researchers interested in harnessing scRNAseq data for deciphering the biology of DCs or other cell types and that it will contribute to establishing high standards in the field., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II - MM were abundant neonatally, whereas MHC-II + MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II + MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

DCs do not just excel in antigen presentation. They orchestrate information transfer from innate to adaptive immunity by sensing and integrating a variety of danger signals, and translating them to naïve T cells, to mount specifically tailored immune responses. This is accomplished by distinct DC types specialized in different functions and because each DC is functionally plastic, assuming different activation states depending on the input signals received. Mouse models hold the key to untangle this complexity and determine which DC types and activation states contribute to which functions. Here, we aim to provide comprehensive information for selecting the most appropriate mutant mouse strains to address specific research questions on DCs, considering three in vivo experimental approaches: (i) interrogating the roles of DC types through their depletion; (ii) determining the underlying mechanisms by specific genetic manipulations; (iii) deciphering the spatiotemporal dynamics of DC responses. We summarize the advantages, caveats, suggested use, and perspectives for a variety of mutant mouse strains, discussing in more detail the most widely used or accurate models. Finally, we discuss innovative strategies to improve targeting specificity and next-generation mutant mouse models, and briefly address how humanized mouse models can accelerate translation into the clinic., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MCOLN1), enabling the local activation of myosin II at the cell rear. Taken together, our findings identify Rab7b as the missing physical link between lysosomes and the actomyosin cytoskeleton, allowing control of immune cell migration through lysosomal signaling. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Successful immune responses rely on a regulated delivery of the right signals to the right cells at the right time. Here we show that natural killer (NK) and dendritic epidermal γδ T cells (DETCs) use similar mechanisms to spatiotemporally orchestrate conventional type 1 dendritic cell (cDC1) functions in the spleen, skin, and its draining lymph nodes (dLNs). Upon MCMV infection in the spleen, cDC1 clusterize with activated NK cells in marginal zones. This XCR1-dependent repositioning of cDC1 toward NK cells allows contact delivery of IL-12 and IL-15/IL-15Rα by cDC1, which is critical for NK cell responses. NK cells deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) to cDC1, guiding their CCR7-dependent relocalization into the T cell zone. In MCMV-infected skin, XCL1-secreting DETCs promote cDC1 migration from the skin to the dLNs. This XCR1-dependent licensing of cDC1 both in the spleen and skin accelerates antiviral CD8 + T cell responses, revealing an additional mechanism through which cDC1 bridge innate and adaptive immunity., Competing Interests: The authors declare no conflict of interests., (© 2021 The Authors.)

Objectives: To better understand how immune responses may be harnessed against breast cancer, we investigated which immune cell types and signalling pathways are required for spontaneous control of a mouse model of mammary adenocarcinoma., Methods: The NOP23 mammary adenocarcinoma cell line expressing epitopes derived from the ovalbumin model antigen is spontaneously controlled when orthotopically engrafted in syngeneic C57BL/6 mice. We combined this breast cancer model with antibody-mediated depletion of lymphocytes and with mutant mice affected in interferon (IFN) or type 1 conventional dendritic cell (cDC1) responses. We monitored tumor growth and immune infiltration including the activation of cognate ovalbumin-specific T cells., Results: Breast cancer immunosurveillance required cDC1, NK/NK T cells, conventional CD4 + T cells and CD8 + cytotoxic T lymphocytes (CTLs). cDC1 were required constitutively, but especially during T-cell priming. In tumors, cDC1 were interacting simultaneously with CD4 + T cells and tumor-specific CTLs. cDC1 expression of the XCR1 chemokine receptor and of the T-cell-attracting or T-cell-activating cytokines CXCL9, IL-12 and IL-15 was dispensable for tumor rejection, whereas IFN responses were necessary, including cDC1-intrinsic signalling by STAT1 and IFN-γ but not type I IFN (IFN-I). cDC1 and IFNs promoted CD4 + and CD8 + T-cell infiltration, terminal differentiation and effector functions. In breast cancer patients, high intratumor expression of genes specific to cDC1, CTLs, CD4 + T cells or IFN responses is associated with a better prognosis., Conclusion: Interferons and cDC1 are critical for breast cancer immunosurveillance. IFN-γ plays a prominent role over IFN-I in licensing cDC1 for efficient T-cell activation., Competing Interests: The authors declare that they have no competing interests., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Currently three bona fide dendritic cell (DC) types are distinguished in human blood. Herein we focus on type 2 DCs (DC2s) and compare the three defining markers CD1c, CD172, and CD301. When using CD1c to define DC2s, a CD14 + and a CD14 - subset can be detected. The CD14 + subset shares features with monocytes, and this includes substantially higher expression levels for CD64, CD115, CD163, and S100A8/9. We review the current knowledge of these CD1c + CD14 + cells as compared to the CD1c + CD14 - cells with respect to phenotype, function, transcriptomics, and ontogeny. Here, we discuss informative mutations, which suggest that two populations have different developmental requirements. In addition, we cover subsets of CD11c + CD8 - DC2s in the mouse, where CLEC12A + ESAM low cells, as compared to the CLEC12A - ESAM high subset, also express higher levels of monocyte-associated markers CD14, CD3, and CD115. Finally, we summarize, for both man and mouse, the data on lower antigen presentation and higher cytokine production in the monocyte-marker expressing DC2 subset, which demonstrate that the DC2 subsets are also functionally distinct., (Copyright © 2020 Heger, Hofer, Bigley, de Vries, Dalod, Dudziak and Ziegler-Heitbrock.)

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.

Dendritic cells (DCs) are mononuclear phagocytes that are specialized in the induction and functional polarization of effector lymphocytes, thus orchestrating immune defenses against infections and cancer. The population of DC encompasses distinct cell types that vary in their efficacy for complementary functions and are thus likely involved in defending the body against different threats. Plasmacytoid DCs specialize in the production of high levels of the antiviral cytokines type I interferons. Type 1 conventional DCs (cDC1s) excel in the activation of cytotoxic CD8 + T cells (CTLs) which are critical for defense against cancer and infections by intracellular pathogens. Type 2 conventional DCs (cDC2s) prime helper CD4 + T cells for the production of type 2 cytokines underpinning immune defenses against worms or of IL-17 promoting control of infections by extracellular bacteria or fungi. Hence, clinically manipulating the development and functions of DC types could have a major impact for improving treatments against many diseases. However, the rarity and fragility of human DC types is impeding advancement towards this goal. To overcome this roadblock, major efforts are ongoing to generate in vitro large numbers of distinct human DC types. We review here the current state of this research field, emphasizing recent breakthrough and proposing future priorities. We also pinpoint the necessity to develop a consensus nomenclature and rigorous methodologies to ensure proper identification and characterization of human DC types. Finally, we elaborate on how faithful in vitro models of human DC types can accelerate our understanding of the biology of these cells and the engineering of next generation vaccines or immunotherapies against viral infections or cancer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 T H 1 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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The monocyte-derived phagocytes termed LysoDCs are hallmarks of Peyer's patches, where their main function is to sample intestinal microorganisms. Here, we study their differentiation pathways in relation with their sampling, migratory, and T cell-priming abilities. Among four identified LysoDC differentiation stages displaying similar phagocytic activity, one is located in follicles, and the others reside in subepithelial domes (SED), where they proliferate and mature as they get closer to the epithelium. Mature LysoDCs but not macrophages express a gene set in common with conventional dendritic cells and prime naive helper T cells in vitro. At steady state, they do not migrate into naive T cell-enriched interfollicular regions (IFRs), but upon stimulation, they express the chemokine receptor CCR7 and migrate from SED to the IFR periphery, where they strongly interact with proliferative immune cells. Finally, we show that LysoDCs populate human Peyer's patches, strengthening their interest as targets for modulating intestinal immunity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell therapy is a promising strategy for treating patients suffering from autoimmune or inflammatory diseases or receiving a transplant. Based on our preclinical studies, we have generated human autologous tolerogenic dendritic cells (ATDCs), which are being tested in a first-in-man clinical trial in kidney transplant recipients. Here, we report that ATDCs represent a unique subset of monocyte-derived cells based on phenotypic, transcriptomic, and metabolic analyses. ATDCs are characterized by their suppression of T cell proliferation and their expansion of Tregs through secreted factors. ATDCs produce high levels of lactate that shape T cell responses toward tolerance. Indeed, T cells take up ATDC-secreted lactate, leading to a decrease of their glycolysis. In vivo, ATDCs promote elevated levels of circulating lactate and delay graft-versus-host disease by reducing T cell proliferative capacity. The suppression of T cell immunity through lactate production by ATDCs is a novel mechanism that distinguishes ATDCs from other cell-based immunotherapies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1 -/- mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7 + stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) are endowed with a unique potency to prime T cells, as well as to orchestrate their expansion, functional polarization and effector activity in non-lymphoid tissues or in their draining lymph nodes. The concept of harnessing DC immunogenicity to induce protective responses in cancer patients was put forward about 25 years ago and has led to a multitude of DC-based vaccine trials. However, until very recently, objective clinical responses were below expectations. Conventional type 1 DCs (cDC1) excel in the activation of cytotoxic lymphocytes including CD8 + T cells (CTLs), natural killer (NK) cells, and NKT cells, which are all critical effector cell types in antitumor immunity. Efforts to investigate whether cDC1 might orchestrate immune defenses against cancer are ongoing, thanks to the recent blossoming of tools allowing their manipulation in vivo . Here we are reporting on these studies. We discuss the mouse models used to genetically deplete or manipulate cDC1, and their main caveats. We present current knowledge on the role of cDC1 in the spontaneous immune rejection of tumors engrafted in syngeneic mouse recipients, as a surrogate model to cancer immunosurveillance, and how this process is promoted by type I interferon (IFN-I) effects on cDC1. We also discuss cDC1 implication in promoting the protective effects of immunotherapies in mouse preclinical models, especially for adoptive cell transfer (ACT) and immune checkpoint blockers (ICB). We elaborate on how to improve this process by in vivo reprogramming of certain cDC1 functions with off-the-shelf compounds. We also summarize and discuss basic research and clinical data supporting the hypothesis that the protective antitumor functions of cDC1 inferred from mouse preclinical models are conserved in humans. This analysis supports potential applicability to cancer patients of the cDC1-targeting adjuvant immunotherapies showing promising results in mouse models. Nonetheless, further investigations on cDC1 and their implications in anti-cancer mechanisms are needed to determine whether they are the missing key that will ultimately help switching cold tumors into therapeutically responsive hot tumors, and how precisely they mediate their protective effects.

Type 1 conventional DCs (cDC1) excel in the cross-priming of CD8 + T cells, which is crucial for orchestrating efficient immune responses against viruses or tumors. However, our understanding of their physiological functions and molecular regulation has been limited by the lack of proper mutant mouse models allowing their conditional genetic targeting. Because the Xcr1 and A530099j19rik ( Karma/Gpr141b) genes belong to the core transcriptomic fingerprint of mouse cDC1, we used them to engineer two novel Cre-driver lines, the Xcr1 Cre and Karma Cre mice, by knocking in an IRES-Cre expression cassette into their 3'-UTR. We used genetic tracing to characterize the specificity and efficiency of these new models in several lymphoid and non-lymphoid tissues, and compared them to the Clec9a Cre mouse model, which targets the immediate precursors of cDCs. Amongst the three Cre-driver mouse models examined, the Xcr1 Cre model was the most efficient and specific for the fate mapping of all cDC1, regardless of the tissues examined. The Karma Cre model was rather specific for cDC1 when compared with the Clec9a Cre mouse, but less efficient than the Xcr1 Cre model. Unexpectedly, the Xcr1 Cre model targeted a small fraction of CD4 + T cells, and the Karma Cre model a significant proportion of mast cells in the skin. Importantly, the targeting specificity of these two mouse models was not changed upon inflammation. A high frequency of germline recombination was observed solely in the Xcr1 Cre mouse model when both the Cre and the floxed alleles were brought by the same gamete irrespective of its gender. Xcr1, Karma , and Clec9a being differentially expressed within the cDC1 population, the three CRE-driver lines examined showed distinct recombination patterns in cDC1 phenotypic subsets. This advances our understanding of cDC1 subset heterogeneity and the differentiation trajectory of these cells. Therefore, to the best of our knowledge, upon informed use, the Xcr1 Cre and Karma Cre mouse models represent the best tools currently reported to specifically and faithfully target cDC1 in vivo , both at steady state and upon inflammation. Future use of these mutant mouse models will undoubtedly boost our understanding of the biology of cDC1.

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., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2018 Baranska et al.)

Cytotoxic CD4 (CD4 CTX ) T cells are emerging as an important component of antiviral and antitumor 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 CD4 CTX -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 CD4 CTX T cells and identifies potential targets for immunotherapy against viral infections and cancer., Competing Interests: YS, CG, BH, MD, TV, AA, AD, AH, JD, MB, EP, TT, EC, Kv, MD, FF, SG, AM No competing interests declared, (© 2018, Serroukh et al.)

The initiation of the mucosal immune response in Peyer's patch (PP) relies on the sampling, processing, and efficient presentation of foreign antigens by dendritic cells (DCs). Among PP DCs, CD11b + conventional DCs (cDCs) and lysozyme-expressing DCs (LysoDCs) have distinct progenitors and functions but share many cell surface markers. This has previously led to confusion between these two subsets. In addition, another PP DC subset, termed double-negative (DN), remains poorly characterized. Here we show that both DN and CD11b + cDCs belong to a unique SIRPα + cDC subset. At steady state, cDCs and TIM-4 + macrophages are mainly located in T-cell zones, i.e., interfollicular regions, whereas a majority of subepithelial phagocytes are monocyte-derived cells, namely, LysoDCs and TIM-4 - macrophages. Finally, oral administration of a Toll-like receptor 7 ligand induces at least three TNF-dependent events: (i) migration of dome-associated villus cDCs in interfollicular regions, (ii) increase of CD8α + interfollicular cDC number, and (iii) activation of both CD11b + and CD8α + interfollicular cDCs. The latter is marked by a genetic reprograming leading to the upregulation of type I interferon-stimulated and of both immuno-stimulatory and -inhibitory gene expression.

Intestinal macrophages (mφ) form one of the largest populations of mφ in the body and are vital for the maintenance of gut homeostasis. They have several unique properties and are derived from local differentiation of classical Ly6C hi monocytes, but the factors driving this tissue-specific process are not understood. Here we have used global transcriptomic analysis to identify a unique homeostatic signature of mature colonic mφ that is acquired as they differentiate in the mucosa. By comparing the analogous monocyte differentiation process found in the dermis, we identify TGFβ as an indispensable part of monocyte differentiation in the intestine and show that it enables mφ to adapt precisely to the requirements of their environment. Importantly, TGFβR signaling on mφ has a crucial role in regulating the accumulation of monocytes in the mucosa, via mechanisms that are distinct from those used by IL10.

In malaria, CD4 Th1 and T follicular helper (T FH ) cells are important for controlling parasite growth, but Th1 cells also contribute to immunopathology. Moreover, various regulatory CD4 T-cell subsets are critical to hamper pathology. Yet the antigen-presenting cells controlling Th functionality, as well as the antigens recognized by CD4 T cells, are largely unknown. Here, we characterize the MHC II immunopeptidome presented by DC during blood-stage malaria in mice. We establish the immunodominance hierarchy of 14 MHC II ligands derived from conserved parasite proteins. Immunodominance is shaped differently whether blood stage is preceded or not by liver stage, but the same ETRAMP-specific dominant response develops in both contexts. In naïve mice and at the onset of cerebral malaria, CD8α + dendritic cells (cDC1) are superior to other DC subsets for MHC II presentation of the ETRAMP epitope. Using in vivo depletion of cDC1, we show that cDC1 promote parasite-specific Th1 cells and inhibit the development of IL-10 + CD4 T cells. This work profiles the P. berghei blood-stage MHC II immunopeptidome, highlights the potency of cDC1 to present malaria antigens on MHC II, and reveals a major role for cDC1 in regulating malaria-specific CD4 T-cell responses., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In lymph nodes (LNs), dendritic cells (DCs) are thought to dispose of apoptotic cells, a function pertaining to macrophages in other tissues. We found that a population of CX3CR1 + MERTK + cells located in the T cell zone of LNs, previously identified as DCs, are efferocytic macrophages. Lineage-tracing experiments and shield chimeras indicated that these T zone macrophages (TZM) are long-lived macrophages seeded in utero and slowly replaced by blood monocytes after birth. Imaging the LNs of mice in which TZM and DCs express different fluorescent proteins revealed that TZM-and not DCs-act as the only professional scavengers, clearing apoptotic cells in the LN T cell zone in a CX3CR1-dependent manner. Furthermore, similar to other macrophages, TZM appear inefficient in priming CD4 T cells. Thus, efferocytosis and T cell activation in the LN are uncoupled processes designated to macrophages and DCs, respectively, with implications to the maintenance of immune homeostasis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.

Dendritic cells (DCs) are critical for the launching of protective T cell immunity in response to viral infection. Viruses can directly infect DCs, thereby compromising their viability and suppressing their ability to activate immune responses. How DC function is maintained in light of this paradox is not understood. By analyzing the susceptibility of primary human DC subsets to viral infections, we report that CD141 + DCs have an innate resistance to infection by a broad range of enveloped viruses, including HIV and influenza virus. In contrast, CD1c + DCs are susceptible to infection, which enables viral antigen production but impairs their immune functions and survival. The ability of CD141 + DCs to resist infection is conferred by RAB15, a vesicle-trafficking protein constitutively expressed in this DC subset. We show that CD141 + DCs rely on viral antigens produced in bystander cells to launch cross-presentation-driven T cell responses. By dissociating viral infection from antigen presentation, this mechanism protects the functional capacity of DCs to launch adaptive immunity against viral infection., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Targeting Ags to conventional dendritic cells can enhance Ag-specific immune responses. Although most studies have focused on the induction of T cell responses, the mechanisms by which targeting improves Ab responses are poorly understood. In this study we present data on the use of human XCL1 (hXCL1) and hXCL2 fusion vaccines in a murine model. We show that the human chemokines bound type 1 conventional dendritic cells (cDC1), and that immunization with influenza virus hemagglutinin fused to hXCL1 or hXCL2 induced full protection against influenza challenge. Surprisingly, the hXCL1- and hXCL2-fusion vaccines induced better long-term protection associated with stronger induction of neutralizing Abs, and more Ab-secreting cells in bone marrow. In contrast, murine Xcl1 fusion vaccines induced stronger CD8 + T cell responses compared with hXCL1. Further analysis revealed that although murine Xcl1 fusion vaccines induced chemotaxis and were rapidly endocytosed by cDC1, hXCL1 and hXCL2 fusion vaccines did not induce chemotaxis, were less efficiently endocytosed, and consequently, remained on the surface. This difference may explain the enhanced induction of Abs when targeting Ag to cDC1 using hXCL1 and hXCL2, and suggests that immune responses can be manipulated in directing Abs or T cells based on how efficiently the targeted Ag is endocytosed by the DC., (Copyright © 2017 by The American Association of Immunologists, Inc.)