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Phagocytosis is a key macrophage function, but how phagocytosis shapes tumor-associated macrophage (TAM) phenotypes and heterogeneity in solid tumors remains unclear. Here, we utilized both syngeneic and novel autochthonous lung tumor models in which neoplastic cells express the fluorophore tdTomato (tdTom) to identify TAMs that have phagocytosed neoplastic cells in vivo. Phagocytic tdTompos TAMs upregulated antigen presentation and anti-inflammatory proteins, but downregulated classic proinflammatory effectors compared to tdTomneg TAMs. Single-cell transcriptomic profiling identified TAM subset-specific and common gene expression changes associated with phagocytosis. We uncover a phagocytic signature that is predominated by oxidative phosphorylation (OXPHOS), ribosomal, and metabolic genes, and this signature correlates with worse clinical outcome in human lung cancer. Expression of OXPHOS proteins, mitochondrial content, and functional utilization of OXPHOS were increased in tdTompos TAMs. tdTompos tumor dendritic cells also display similar metabolic changes. Our identification of phagocytic TAMs as a distinct myeloid cell state links phagocytosis of neoplastic cells in vivo with OXPHOS and tumor-promoting phenotypes.

Conventional type 1 dendritic cells (cDC1)1 are thought to perform antigen cross-presentation, which is required to prime CD8+ T cells2,3, whereas cDC2 are specialized for priming CD4+ T cells4,5. CD4+ T cells are also considered to help CD8+ T cell responses through a variety of mechanisms6–11, including a process whereby CD4+ T cells ‘license’ cDC1 for CD8+ T cell priming12. However, this model has not been directly tested in vivo or in the setting of help-dependent tumour rejection. Here we generated an Xcr1Cre mouse strain to evaluate the cellular interactions that mediate tumour rejection in a model requiring CD4+ and CD8+ T cells. As expected, tumour rejection required cDC1 and CD8+ T cell priming required the expression of major histocompatibility class I molecules by cDC1. Unexpectedly, early priming of CD4+ T cells against tumour-derived antigens also required cDC1, and this was not simply because they transport antigens to lymph nodes for processing by cDC2, as selective deletion of major histocompatibility class II molecules in cDC1 also prevented early CD4+ T cell priming. Furthermore, deletion of either major histocompatibility class II or CD40 in cDC1 impaired tumour rejection, consistent with a role for cognate CD4+ T cell interactions and CD40 signalling in cDC1 licensing. Finally, CD40 signalling in cDC1 was critical not only for CD8+ T cell priming, but also for initial CD4+ T cell activation. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of antigen processing and priming for both CD4+ and CD8+ T cells and of the direct orchestration of their cross-talk that is required for optimal anti-tumour immunity. Conventional type 1 dendritic cells perform antigen processing and priming of CD4+ and CD8+ T cells against tumour antigens, orchestrating their cross-talk to effect anti-tumour immunity.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7sup+/supcDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification. The transcription factor IRF8 is essential for classical type 1 dendritic cell (cDC1) development. Murphy and colleagues investigate in detail the molecular control of cDC1 fate specification by systematically unpicking the IRF8 enhancer regions.

The BATF3-dependent cDC1 lineage of conventional dendritic cells (cDC) is required for rejection of immunogenic sarcomas and for rejection of progressive sarcomas during checkpoint blockade therapy. One unique function of the cDC1 lineage is the efficient cross-presentation of tumor-derived neoantigens to CD8+ T cells, but it is not clear that this is the only unique function of cDC1 required for tumor rejection. We previously showed that BATF3 functions during cDC1 lineage commitment to maintain IRF8 expression in the specified cDC1 progenitor. However, since cDC1 progenitors do not develop into mature cDC1s in Batf3−/− mice, it is still unclear whether BATF3 has additional functions in mature cDC1 cells. A transgenic Irf8-Venus reporter allele increases IRF8 protein concentration sufficiently to allow autonomous cDC1 development in spleens of Batf3−/− mice. These restored Batf3−/− cDC1s are transcriptionally similar to control wild-type cDC1s but have reduced expression of a restricted set of cDC1-specific genes. Restored Batf3−/− cDC1s are able to cross-present cell-associated antigens both in vitro and in vivo. However, Batf3−/− cDC1 exhibit altered characteristics in vivo and are unable to mediate tumor rejection. These results show that BATF3, in addition to regulating Irf8 expression to stabilize cDC1 lineage commitment, also controls expression of a small set of genes required for cDC1-mediated tumor rejection. These BATF3-regulated genes may be useful targets in immunotherapies aimed at promoting tumor rejection.

Adding to the cross-presentation family Immune responses to viral or tumor antigens are typically initiated by the process of cross-presentation. Cross-presentation is believed to be the major way that innate immune cells, such as the classical dendritic cell 1 (cDC1) subset, activate and prime immunological T cells. Theisen et al. used CRISPR-based screening to identify regulators of cross-presentation by cDC1s (see the Perspective by Barbet and Blander). One such regulator that was identified, WDFY4 (WD repeat- and FYVE domain–containing protein 4), was required for cross-presentation of cell- and bacterial-associated antigens. WDFY4 played a critical role in cDC1-mediated viral and tumor immunity yet did not seem necessary for major histocompatibility complex class II presentation or for cross-presentation by monocyte-derived DCs. Science , this issue p. 694 ; see also p. 641

Significance High-affinity antibody responses involve selection of B cells in the germinal center (GC) by cognate interactions with T follicular helper (TFH) cells, which in turn must first be activated by classical dendritic cells (cDCs). We observe that Notch2-dependent cDC2s are required in vivo for induction of TFH cells, GC B cells, and specific antibody production in response to sheep red blood cell (SRBC) immunization. Notch2 signaling impacted a broad transcriptional program in cDC2s both at homeostasis and after SRBC immunization, although we have not identified a target gene that mediates TFH differentiation. Thus, Notch2 is a transcription factor that acts in cDCs and is selectively required for support of the GC reaction., CD4+ T follicular helper (TFH) cells support germinal center (GC) reactions promoting humoral immunity. Dendritic cell (DC) diversification into genetically distinct subsets allows for specialization in promoting responses against several types of pathogens. Whether any classical DC (cDC) subset is required for humoral immunity is unknown, however. We tested several genetic models that selectively ablate distinct DC subsets in mice for their impact on splenic GC reactions. We identified a requirement for Notch2-dependent cDC2s, but not Batf3-dependent cDC1s or Klf4-dependent cDC2s, in promoting TFH and GC B cell formation in response to sheep red blood cells and inactivated Listeria monocytogenes. This effect was mediated independent of Il2ra and several Notch2-dependent genes expressed in cDC2s, including Stat4 and Havcr2. Notch2 signaling during cDC2 development also substantially reduced the efficiency of cDC2s for presentation of MHC class II-restricted antigens, limiting the strength of CD4 T cell activation. Together, these results demonstrate a nonredundant role for the Notch2-dependent cDC2 subset in supporting humoral immune responses.

Conventional type 1 dendritic cells (cDC1)

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow–resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box–binding protein associated with epithelial–mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6Chi monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals.

RAB43 is a vesicular transport protein unique to CD8α+ DCs that is localized to the Golgi. Kretzer et al. show that RAB43 is necessary for optimal cross-presentation of cell-associated antigens by CD8α+ DCs in vitro and in vivo but that it is dispensable for cross-presentation by in vitro monocyte-derived DCs., In this study, to examine cross-presentation by classical dendritic cells (DCs; cDCs), we evaluated the role of RAB43, a protein found to be selectively expressed by Batf3-dependent CD8α+ and CD103+ compared with other DC subsets and immune lineages. Using a specific monoclonal antibody, we localized RAB43 expression to the Golgi apparatus and LAMP1− cytoplasmic vesicles. Mice with germline or conditional deletion of Rab43 are viable and fertile and have normal development of cDCs but show a defect for in vivo and in vitro cross-presentation of cell-associated antigen. This defect is specific to cDCs, as Rab43-deficient monocyte-derived DCs showed no defect in cross-presentation of cell-associated antigen. These results suggest that RAB43 provides a specialized activity used in cross-presentation selectively by CD8α+ DCs but not other antigen-presenting cells.

The dendritic cells (DCs) of the immune system function in innate and adaptive responses by directing activity of various effector cells rather than serving as effectors themselves. DCs and closely related myeloid lineages share expression of many surface receptors, presenting a challenge in distinguishing their unique in vivo functions. Recent work has taken advantage of unique transcriptional programs to identify and manipulate murine DCs in vivo. This work has assigned several nonredundant in vivo functions to distinct DC lineages, consisting of plasmacytoid DCs and several subsets of classical DCs that promote different immune effector modules in response to pathogens. In parallel, a correspondence between human and murine DC subsets has emerged, underlying structural similarities for the DC lineages between these species. Recent work has begun to unravel the transcriptional circuitry that controls the development and diversification of DCs from common progenitors in the bone marrow.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor alpha (VEGF-alpha) by a minor subset of activated EpCAM(+)CD59(+) Ly-6D(+) cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guerin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Classical type 1 dendritic cells (cDC1s) are required for antiviral and antitumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E-protein-dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor Irf8 (+41-kb Irf8 enhancer), but its maturation instead requires the Batf3-dependent +32-kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E-protein activity to exclude plasmacytoid dendritic cell potential and explains the switch in Irf8 enhancer usage during cDC1 development. Classical type 1 dendritic cells (cDC1s) are essential for activation of antiviral and anticancer T cell responses. Murphy and colleagues describe a genetic circuit that involves the transcription factors Nfil3, Id2 and Zeb2. This circuit imposes a molecular switch that allows cDC1 specification and development.

The receptor Flt3 and its ligand Flt3L are both critical for dendritic cell (DC) development, but DC deficiency is more severe in Flt3l−/− mice than in Flt3−/− mice. This has led to speculation that Flt3L binds to another receptor that also supports DC development. However, we found that Flt3L administration does not generate DCs in Flt3−/− mice, arguing against a second receptor. Instead, Flt3−/− DC progenitors matured in response to macrophage colony–stimulating factor (M-CSF) or stem cell factor, and deletion of Csf1r in Flt3−/− mice further reduced DC development, indicating that these cytokines could compensate for Flt3. Surprisingly, Flt3−/− DC progenitors displayed enhanced M-CSF signaling, suggesting that loss of Flt3 increased responsiveness to other cytokines. In agreement, deletion of Flt3 in Flt3l−/− mice paradoxically rescued their severe DC deficiency. Thus, multiple cytokines can support DC development, and the discrepancy between Flt3−/− and Flt3l−/− mice results from the increased sensitivity of Flt3−/− progenitors to these cytokines.

The study of murine dendritic cell (DC) development has been integral to the identification of specialized DC subsets that have unique requirements for their form and function. Advances in the field have also provided a framework for the identification of human DC counterparts, which appear to have conserved mechanisms of development and function. Multiple transcription factors are expressed in unique combinations that direct the development of classical DCs (cDCs), which include two major subsets known as cDC1s and cDC2s, and plasmacytoid DCs (pDCs). pDCs are potent producers of type I interferons and thus these cells are implicated in immune responses that depend on this cytokine. Mouse models deficient in the cDC1 lineage have revealed their importance in directing immune responses to intracellular bacteria, viruses, and cancer through the cross-presentation of cell-associated antigen. Models of transcription factor deficiency have been used to identify subsets of cDC2 that are required for T helper (Th)2 and Th17 responses to certain pathogens; however, no single factor is known to be absolutely required for the development of the complete cDC2 lineage. In this review, we will discuss the current state of knowledge of mouse and human DC development and function and highlight areas in the field that remain unresolved.

Durai et al. demonstrate that the less severe DC deficiency in Flt3–/– mice compared to Flt3l–/– mice is due to the enhanced sensitivity of Flt3–/– progenitors to compensatory cytokines that support DC development., The receptor Flt3 and its ligand Flt3L are both critical for dendritic cell (DC) development, but DC deficiency is more severe in Flt3l−/− mice than in Flt3−/− mice. This has led to speculation that Flt3L binds to another receptor that also supports DC development. However, we found that Flt3L administration does not generate DCs in Flt3−/− mice, arguing against a second receptor. Instead, Flt3−/− DC progenitors matured in response to macrophage colony–stimulating factor (M-CSF) or stem cell factor, and deletion of Csf1r in Flt3−/− mice further reduced DC development, indicating that these cytokines could compensate for Flt3. Surprisingly, Flt3−/− DC progenitors displayed enhanced M-CSF signaling, suggesting that loss of Flt3 increased responsiveness to other cytokines. In agreement, deletion of Flt3 in Flt3l−/− mice paradoxically rescued their severe DC deficiency. Thus, multiple cytokines can support DC development, and the discrepancy between Flt3−/− and Flt3l−/− mice results from the increased sensitivity of Flt3−/− progenitors to these cytokines.

RelB is an NF-κB family transcription factor activated in the noncanonical pathway downstream of NF-κB-inducing kinase (NIK) and TNF receptor family members including lymphotoxin-β receptor (LTβR) and CD40. Early analysis suggested that RelB is required for classical dendritic cell (cDC) development based on a severe reduction of cDCs in Relb-/- mice associated with profound myeloid expansion and perturbations in B and T cells. Subsequent analysis of radiation chimeras generated from wild-type and Relb-/- bone marrow showed that RelB exerts cell-extrinsic actions on some lineages, but it has remained unclear whether the impact of RelB on cDC development is cell-intrinsic or -extrinsic. Here, we reevaluated the role of RelB in cDC and myeloid development using a series of radiation chimeras. We found that there was no cell-intrinsic requirement for RelB for development of most cDC subsets, except for the Notch2- and LTβR-dependent subset of splenic CD4+ cDC2s. These results identify a relatively restricted role of RelB in DC development. Moreover, the myeloid expansion in Relb-/- mice resulted from hematopoietic-extrinsic actions of RelB. This result suggests that there is an unrecognized but critical role for RelB within the nonhematopoietic niche that controls normal myelopoiesis.

Cross-presentation allows for the presentation of exogenous viral and tumor antigens to CD8+ T cells by XCR1+ classical dendritic cells (cDC1s). Previously, the role of cross-presentation in cytotoxic T cell responses in vivo was unclear due to the lack of mouse models specifically deficient in cross-presentation. We developed a CRISPR-Cas9 screen to find novel regulators of cross-presentation and found that the protein WDFY4 is essential for cross-presentation of cell-associated antigens but not for direct presentation or presentation on MHCII. WDFY4-deficient mice had normal cDC1 development and were able to survive infections with Toxoplasma gondii through production of IL-12. However, WDFY4-deficient mice failed to prime CD8+ T cells to viral antigens and were completely unable to control immunogenic tumors. WDFY4 interacts with proteins related to vesicular formation and trafficking such as clathrin and HSP90ab1 and localizes near the cell surface, suggesting its involvement in vesicular targeting after antigen uptake during cross-presentation.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.

Variable strengths of T cell receptor (TCR) signaling can produce divergent outcomes, but the mechanism remains obscure. The abundance of the transcription factor IRF4 increases with TCR signal strength, but how this would induce distinct types of responses is unclear. We compared TH2 gene expression with BATF/IRF4 enhancer occupancy at varying strengths of TCR stimulation. BATF/IRF4-dependent genes clustered into distinct TCR-sensitivities. Enhancers exhibited a spectrum of occupancy by BATF/IRF4 ternary complex that correlated with TCR-sensitivity of gene expression. DNA sequences immediately flanking the previously defined AICE motif controlled the affinity for BATF/IRF4 for direct binding to DNA. ChIP-exo analysis allowed identification of a novel high-affinity AICE2 motif at a human SNP of CTLA4 associated with resistance to autoimmunity. Thus, the affinity of different enhancers for the BATF-IRF4 complex may underlie divergent signaling outcomes in response to various strengths of TCR signaling.

Using Mafb-driven Cre, Murphy et al. establish a new tool to discriminate macrophages from other myeloid cells in vivo., Current systems for conditional gene deletion within mouse macrophage lineages are limited by ectopic activity or low efficiency. In this study, we generated a Mafb-driven Cre strain to determine whether any dendritic cells (DCs) identified by Zbtb46-GFP expression originate from a Mafb-expressing population. Lineage tracing distinguished macrophages from classical DCs, neutrophils, and B cells in all organs examined. At steady state, Langerhans cells (LCs) were lineage traced but also expressed Zbtb46-GFP, a phenotype not observed in any other population. After exposure to house dust mite antigen, Zbtb46-negative CD64+ inflammatory cells infiltrating the lung were substantially lineage traced, but Zbtb46-positive CD64− cells were not. These results provide new evidence for the unique identity of LCs and challenge the notion that some inflammatory cells are a population of monocyte-derived DCs.

International audience; Lymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in Ccr7–/– mice is followed by vessel fibrosis. Reexpression of CCR7 in DCs, however, was sufficient to reverse the development of such fibrosis. IFN regulatory factor 4–positive (IRF4+) DCs constitutively interacted with collecting lymphatics, and selective ablation of this DC subset in Cd11c-Cre Irf4fl/fl mice also rendered lymphatic collecting vessels hyperpermeable and fibrotic. Together, our data reveal that CCR7 plays multifaceted roles in regulating collecting vessel permeability and fibrosis, with one of the key players being IRF4-dependent DCs.

Introduction: Dendritic cells (DCs) are professional antigen-presenting cells with two major subsets, the classical DC type 1 and 2 (cDC1 and cDC2). The cDC1, also known as the CD8α+ DC, is especially equipped for the process of cross-presentation in which dead cell antigen is presented on MHC-I to prime cytotoxic T cells. This process is relevant to anti-cancer immunity, as mice deficient in the transcription factor BATF3, which specifically lack the cDC1, succumb to highly immunogenic tumors that are normally rejected and cleared in wild-type mice. The mechanism of cross-presentation by the cDC1 is poorly understood. In this study, we developed a focused CRISPR/Cas9 screen to identify novel genes that endow the cDC1 with the ability to cross-prime cytotoxic T cells. Methods: A curated list of target cDC1 genes was constructed using the Immunological Genome Project Database as the primary resource (www.immgen.org). 120 genes specifically expressed by the cDC1 in comparison to other murine immune cells were selected. Guide RNAs (gRNAs) were designed to early exon sequences using the MIT CRISPR Design Tool to minimize off-target effects (www.crispr.mit.edu), and 2-3 different gRNAs were evaluated in tandem for each candidate. These gRNAs were transduced via retroviral vector into lineage depleted c-Kithi bone marrow cells isolated from C57BL/6J Cas9 knock-in mice. These cells were differentiated in vitro for 8 days in the presence of the cytokine Flt3L. Transduced DCs were sorted by FACS and co-cultured in the presence of a source of dead cell antigen (heat-killed L. monocytogenes expressing ovalbumin, or HKLM-OVA) as well as CFSE-labelled transgenic CD8+ T cells with an ovalbumin peptide-specific T cell receptor (OT-1). The proliferation of OT-1 cells was measured by CFSE dilution. Results: To validate our CRISPR/Cas9-based screening approach, we targeted the first exon of the beta-2 microglobulin gene (B2M), which is required for cell surface expression of MHC-I. B2M knockout DCs were co-cultured in the presence of HKLM-OVA and CSFE-labelled OT-1 cells for 3 days. Controls included DCs transduced with a scramble gRNA with or without antigen. As expected, targeting B2M in DCs led to near-complete reduction in OT-1 proliferation compared to scramble control (3.7% vs. 20.6%, p=0.003), similar to the absence of antigen (3.7% vs. 0.96%, p=0.99). We then expanded our assay to our curated list. Promising candidate genes have been discovered and are currently being validated in murine knockout models. Conclusions: The CRISPR/Cas9 system can be adapted to screen the cDC1 for novel genes involved in cross-priming cytotoxic T cells. Validation of candidates in vivo, including tumor assays, will be presented at the meeting. These findings may have important implications for adaptive immunity against cancer. Citation Format: Jesse T. Davidson, Derek J. Theisen, Carlos G. Briseño, Vivek Durai, William E. Gillanders, Theresa L. Murphy, Kenneth M. Murphy. A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3781.

SUMMARY Although the outcome of flavivirus infection can vary from asymptomatic to lethal, environmental factors modulating disease severity are poorly defined. Here, we observed increased susceptibility of mice to severe West Nile (WNV), Dengue, and Zika virus infections after treatment with oral antibiotics (Abx) that depleted the gut microbiota. Abx treatment impaired the development of optimal T cell responses, with decreased levels of WNV-specific CD8+ T cells associated with increased infection and immunopathology. Abx treatments that resulted in enhanced WNV susceptibility generated changes in the overall structure of the gut bacterial community and in the abundance of specific bacterial taxa. As little as 3 days of treatment with ampicillin was sufficient to alter host immunity and WNV outcome. Our results identify oral Abx therapy as a potential environmental determinant of systemic viral disease, and they raise the possibility that perturbation of the gut microbiota may have deleterious consequences for subsequent flavivirus infections., In Brief Thackray et al. observed increased susceptibility to West Nile, Zika, and Dengue virus infections following oral antibiotic treatment in mice. Antibiotics altered the bacterial abundance and community structure and the development of optimal T cell immunity. These data suggest that antibiotics may have deleterious consequences for subsequent flavivirus infections.

Nature Communications 7: Article number: 12597 (2016); Published: 31 August 2016; Updated: 3 May 2017 The authors inadvertently omitted Christine Häger, who was involved in the initial characterization of Notch mutant mice presented in this Article, from the author list and Author contributions statement.

The transcription factors Batf3 and IRF8 are required for the development of CD8α(+) conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α(+) cDCs and CD4(+) cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α(+) cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α(+) cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3(-/-) mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α(+) cDCs due to decay of Irf8 autoactivation and diverted to the CD4(+) cDC lineage.

Runx1 and Cbfβ are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbfβ is essential for the development of Flt3(+) macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105(+) erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbfβ restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia.

Dendritic cells (DCs) are professional antigen presenting cells conventionally thought to mediate cellular adaptive immune responses. Recent studies have led to the recognition of a non-redundant role for DCs in orchestrating innate immune responses, and in particular, for DC subset-specific interactions with innate lymphoid cells (ILCs). Recently recognized as important effectors of early immune responses, ILCs develop into subsets which mirror the transcriptional and cytokine profile of their T cell subset counterparts. DC diversification into functional subsets provides for modules of pathogen sensing and cytokine production that direct pathogen-appropriate ILC and T cell responses. This review focuses on the recent advances in the understanding of DC development, and their function in orchestrating the innate immune modules.

Recent work has shown that the transcription factor Zeb2 is regulated by and cooperates with T-bet to promote terminal differentiation of NK cells and cytotoxic T cells. In the mouse, Zeb2 is expressed throughout dendritic cell (DC) development and in mature plasmacytoid DCs (pDCs) and CD11b+ classical DCs (cDCs), but not in CD8+/CD103+ cDCs. We hypothesized that downregulation of Zeb2 expression would be essential for the development of CD8+/CD103+ cDCs. Upon conditional deletion of Zeb2, we observed a substantial decrease in pDC frequency and an increased ratio of CD8+:CD11b+ cDCs; meanwhile, Zeb2 haploinsufficiency yielded a partial defect in pDC development. Additional experiments demonstrated that this defect was cell-intrinsic and durable, and that it could not be rescued by administration of type I interferon, which increased pDC frequency in cultures of wild-type DC progenitors but directed Zeb2-deficient DC progenitors almost entirely to the CD8+/CD103+ cDC lineage. Deletion of Zeb2 induced by poly(I:C) treatment in an Mx1-Cre-driven model also resulted in perturbations in other myeloid lineages. Although poly(I:C) treatment led to an acute loss of Ly-6Chi monocytes both in Zeb2fl/fl;Mx1-Cre(tg) mice and in control mice, those monocytes were replenished to baseline frequencies in control mice but not in Zeb2-deficient mice. Similar decreases in monocyte frequency were observed in other organs, and further experiments suggested that this defect was also cell-intrinsic, gene dosage-dependent, durable, and observable in model systems that do not require use of poly(I:C). Thus, we have identified an essential regulator of development in multiple myeloid lineages.

The transcription factors c-Myc and N-Myc--encoded by Myc and Mycn, respectively--regulate cellular growth and are required for embryonic development. A third paralogue, Mycl1, is dispensable for normal embryonic development but its biological function has remained unclear. To examine the in vivo function of Mycl1 in mice, we generated an inactivating Mycl1(gfp) allele that also reports Mycl1 expression. We find that Mycl1 is selectively expressed in dendritic cells (DCs) of the immune system and controlled by IRF8, and that during DC development, Mycl1 expression is initiated in the common DC progenitor concurrent with reduction in c-Myc expression. Mature DCs lack expression of c-Myc and N-Myc but maintain L-Myc expression even in the presence of inflammatory signals such as granulocyte-macrophage colony-stimulating factor. All DC subsets develop in Mycl1-deficient mice, but some subsets such as migratory CD103(+) conventional DCs in the lung and liver are greatly reduced at steady state. Importantly, loss of L-Myc by DCs causes a significant decrease in in vivo T-cell priming during infection by Listeria monocytogenes and vesicular stomatitis virus. The replacement of c-Myc by L-Myc in immature DCs may provide for Myc transcriptional activity in the setting of inflammation that is required for optimal T-cell priming.

SummarySplenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80+VCAM1+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.