Common DC Progenitors Research Articles

Anatomy of a superenhancer.

Interferon regulatory factor-8 (IRF8) is the lineage determining transcription factor for the type one classical dendritic cell (cDC1) subset, a terminal selector for plasmacytoid dendritic cellsand important for the function of monocytes. Studies of Irf8 gene regulation have identified several enhancers controlling its activity during development of progenitors in the bone marrowthat precisely regulate expression at distinct developmental stages. Each enhancer responds to distinct transcription factors that are expressed at each stage. IRF8 is first expressed in early progenitors that form the monocyte dendritic cell progenitor (MDP) in response to induction of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) acting at the Irf8 +56kb enhancer. IRF8 levels increase further as the MDP transits into the common dendritic cell progenitor (CDP) in response to E protein activity at the Irf8 +41kb enhancer. Upon Nfil3-induction in CDPs leading to specification of the cDC1 progenitor, abrupt induction of BATF3 forms the JUN/BATF3/IRF8 heterotrimer that activates the Irf8 +32kb enhancer that sustains Irf8 autoactivation throughout the cDC1 lifetime. Deletions of each of these enhancers has revealed their stage dependent activation. Surprisingly, studies of compound heterozygotes for each combination of enhancer deletions revealed that activation of each subsequent enhancer requires the successful activation of the previous enhancer in strictly cis-dependent mechanism. Successful progression of enhancer activation is finely tuned to alter the functional accessibility of subsequent enhancers to factors active in the next stage of development. The molecular basis for these phenomenon is still obscure but could have implications for genomic regulation in a broader developmental context.

Aberrant monocytopoiesis drives granuloma development in sarcoidosis.

In sarcoidosis, granulomas develop in multiple organs including the liver and lungs. Although mechanistic target of rapamycin complex 1 (mTORC1) activation in macrophages drives granuloma development in sarcoidosis by enhancing macrophage proliferation, little is known about the macrophage subsets that proliferate and mature into granuloma macrophages. Here, we show that aberrantly increased monocytopoiesis gives rise to granulomas in a sarcoidosis model, in which Tsc2, a negative regulator of mTORC1, is conditionally deleted in CSF1R-expressing macrophages (Tsc2csf1rΔ mice). In Tsc2csf1rΔ mice, common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), common monocyte progenitors / monocyte progenitors (cMoPs / MPs), inducible monocyte progenitors (iMoPs), and Ly6Cint CX3CR1low CD14- immature monocytes (iMOs), but not monocyte-dendritic cell progenitors (MDPs) and common dendritic cell progenitors (CDPs), accumulated and proliferated in the spleen. Consistent with this, monocytes, neutrophils, and neutrophil-like monocytes increased in the spleens of Tsc2csf1rΔ mice, whereas dendritic cells did not. The adoptive transfer of splenic iMOs into wild-type mice gave rise to granulomas in the liver and lungs. In these target organs, iMOs matured into Ly6Chi classical monocytes/macrophages (cMOs). Giant macrophages (gMAs) also accumulated in the liver and lungs, which were similar to granuloma macrophages in expression of cell surface markers such as MerTK and SLAMF7. Furthermore, the gMA-specific genes were expressed in human macrophages from sarcoidosis skin lesions. These results suggest that mTORC1 drives granuloma development by promoting the proliferation of monocyte/neutrophil progenitors and iMOs predominantly in the spleen, and that proliferating iMOs mature into cMOs and then gMAs to give rise to granuloma after migration into the liver and lungs in sarcoidosis.

Identification of dendritic cell precursor from the CD11c+ cells expressing high levels of MHC class II molecules in the culture of bone marrow with FLT3 ligand.

Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.

A Landscape of Dendritic Cells Development, Activation and Migration in Mouse Model of Sickle Cell Anemia

Dendritic cells (DCs) are crucial players of innate immunity in initiating, polarizing, and regulating the adaptive immune response. They become activated upon encountering either pathogen- or damage-associated molecular patterns (PAMPs or DAMPs), which are often released due to tissue necrosis or intravascular hemolysis, common events in individuals with sickle cell disease (SCD), one of the most common hemoglobinophaties that affects millions of people worldwide. Upon activation, DCs migrate to peripheral lymph nodes, where they present antigens to T lymphocyte together with co-stimulatory or inhibitory molecules. In addition, they can polarize T cell response by releasing pro- or anti-inflammatory cytokines. We previously showed that SCD patients have higher circulating total and inflammatory DCs, whereas the ratio of type 1 conventional DCs (cDC1) is lower than control individuals. These changes correlated with number of reticulocytes and with the skewed T response towards Th17. Alterations in the development and activation of DC subpopulations, as well as in their function can impair the immune response or tolerance, which may account for the dysfunction in T lymphocyte responses observed in SCD patients. In order to investigate the development, activation and migration of DCs during SCD, we characterized these cells by flow cytometry in lymphoid organs of Townes mice, which have human S hemoglobin (HbS). Our data show that Townes mice (SS) have lower number of total DCs in bone marrow (p=0.057; N=8 each), however, they have higher number of these cells in the spleen (p<0.0001; N=8 each) and lymph nodes (p<0.0021; N=8 each) when compared with the littermate WT mice, suggesting migration of DCs to these peripheral organs. Evaluation of the ratios of DCs subsets shows that the frequency of cDC1 (CD11c +CD8a +CD11b -) among total DCs is higher in the three organs of SS mice compared with WT mice (p=0.004 in spleen; p=0.0015 in bone marrow; and p=0.01 in lymph nodes; N=8 each group). In contrast, the percentage of type 2 conventional DCs, cDC2 (CD11c +CD8a -CD11b +) is similar or lower in SS mice than WT mice (p=0.0005 in spleen; N=8 each). In addition, the frequency of plasmacytoid dendritic cell, pDC (CD11c +B220 +SiglecH +) is higher in lymph nodes (p=0.0006; N=8 each) and lower in bone marrow (p=0.0017; N=8 each) of SS mice when compared with WT mice, indicating that the migration of DCs to peripheral organs is due to cDC1 and pDC. Regarding their maturation and activation phenotype, cDC1 and cDC2 from SS mice have a more immature or inhibitory phenotype than WT mice, as showed by MHC-II (cDC1: p=0.01 in spleen; p=0.0004 in lymph nodes; N=8 each) and CD86 expression (cDC1: p=0.013 in spleen; p=0.0003 in bone marrow; and p= 0.03 in lymph nodes; cDC2: p=0.0067 in bone marrow and p=0.038 in lymph nodes; N=8 each). On the other hand, pDCs from the three organs of SS mice showed a more mature profile than WT mice, as demonstrated by an increase in Sca-1 expression (p=0.0024 in spleen; p=0.037 in bone marrow; and p=0.04 in lymph nodes; N=8 each), indicating that cDCs are being inhibited whereas pDCs are activated during SCD even in steady state. Aimed to understand whether at least part of the alterations on DCs is due to changes in their development, we quantified monocyte and DC progenitor, MDP (Lin -(CD3/CD19/CD56/CD14) CD135 +CD11c -CD115 +CD117 +) and common DC progenitor, CDP (Lin -CD135 +CD11c -CD115 +CD117 -) in bone barrow. Our data show that the ratio of MDP, the earlier progenitor, is increased (p=0.0009; N=8 each), whereas CDP, the progenitor committed to DCs, is diminished (p=0.014; N=8 each) in SS mice when compared with WT mice, indicating a change in the DC compartment in SS mice since their developmental stage. When we performed an in vitro differentiation of DC from bone marrow cells in the presence of flt3l, we did not observe statistical difference in either development and apoptosis of DCs, or in the percentages of DC subsets between SS and WT mice, indicating that the alteration in DC development in SS mice may be due to environmental conditions instead of CDP or DC intrinsic factor. Our findings so far improve our understanding of DCs dynamics in SCD, including the development, activation and migration of the different subsets that may have an important impact on adaptive immune response disfunction in SCD, which may account for the chronic inflammatory state and the susceptibility to infections.

A Multimodal Single Cell Atlas of Adult Murine Haematopoiesis Reveals Nine Differentiation Trajectories and Epigenetic Regulatory Programs

Single cell genomics has revolutionised our understanding of differentiating systems and led to a reinterpretation of haematopoiesis, moving from a step-wise hierarchical tree model towards more continous differentiation landscapes. Recent single cell RNA sequencing (scRNA-seq) studies of Lineage- ckit+ (LK) mouse bone marrow progenitors have defined a highly granular landscape of differentiation from multipotent stem cells to committed progenitors in 8 different lineages and have served as a valuable reference landscape for comparisons with perturbation states. While these landscapes have permitted exploration and interrogation of early haematopoietic progenitors, as yet, no map of mouse total bone marrow haematopoiesis exists at single cell resolution, limiting efforts to fully understand the gene programs which specify and the cell surface phenotypes which identify maturing haematopoietic lineages nor the connection between early bifurcation decisions in progenitor cells and mature cellular outputs. To answer these questions, we performed droplet-based scRNA-seq and cell surface proteomics on whole bone marrow from 4 male and 4 female 12-week old C57Bl6 mice, using a panel of 138 antibodies against cell surface antigens. To increase representation of rare progenitor populations, we complemented total bone marrow mononuclear cells with c-kit enrichment and FACS sorted LK and LK sca1-positive (LSK) populations. After quality control filtering the single cell transcriptomes and proteomes, over 198,000 single cells were analysed separately before multimodal integration into a single UMAP embedding. Addition of cell surface proteome information to the scRNA-seq data through multimodal integration produced a representation with greater resolution of haematopoietic cell types, including improved resolution of T-cell subtypes and separation of a rare population of eosinophil progenitors which were previously indistinguishable from basophil progenitors. This integrated landscape of mouse haematopoiesis was then clustered and annotated though an iterative process of label transfer and manual annotation into 41 cell type states (Figure 1). Using cell fate analysis, which utilises transcriptional similarity and pseudotemporal ordering to estimate the probability of a single cell's commitment to defined terminal states, we were able to resolve cell fate probabilities towards nine haematopoietic lineages (erythroid, neutrophil, megakaryocyte, lymphoid, monocyte, dendritic cell (DC), plasmacytoid dendritic cell (pDC), basophil and eosinophil (Figure 2)). From these probabilities we defined nine trajectories from the earliest uncommitted progenitors to terminally differentiated populations, including a pDC trajectory involving both lymphoid and common DC progenitors. Analysis of trajectory-based gene expression trends provides a framework for discovery of lineage drivers as well as shared gene expression programs across multiple lineages. Leveraging cell surface proteome data permits the use of ‘in-silico-FACS’ to describe phenotypes of cell populations which exist at cell fate branch-points, for further in-vitro characterisation. To demonstrate the utility of our atlas as a reference, we projected perturbation datasets from 7 pre-leukaemic mouse models onto the atlas, allowing quantitation of mutation-associated cellular and tissue-scale alterations. Finally, we have leveraged the transcription-start site enrichment of 5' scRNA-seq to compute enhancer RNA (eRNA) expression in our atlas, allowing the identification of dynamic usage of specific enhancer elements during haematopoietic specification and thus providing a novel framework for correlation of eRNA expression (as a proxy for active distal chromatin regions) with gene expression trends along specific haematopoietic lineages.

Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

IL-6 selectively suppresses cDC1 specification via C/EBPβ.

Cytokines produced in association with tumors can impair antitumor immune responses by reducing the abundance of type 1 conventional dendritic cells (cDC1), but the mechanism remains unclear. Here, we show that tumor-derived IL-6 generally reduces cDC development but selectively impairs cDC1 development in both murine and human systems through the induction of C/EBPβ in the common dendritic cell progenitor (CDP). C/EBPβ and NFIL3 compete for binding to sites in the Zeb2 -165 kb enhancer and support or repress Zeb2 expression, respectively. At homeostasis, pre-cDC1 specification occurs upon Nfil3 induction and consequent Zeb2 suppression. However, IL-6 strongly induces C/EBPβ expression in CDPs. Importantly, the ability of IL-6 to impair cDC development is dependent on the presence of C/EBPβ binding sites in the Zeb2 -165 kb enhancer, as this effect is lost in Δ1+2+3 mutant mice in which these binding sites are mutated. These results explain how tumor-associated IL-6 suppresses cDC1 development and suggest therapeutic approaches preventing abnormal C/EBPβ induction in CDPs may help reestablish cDC1 development to enhance antitumor immunity.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-β, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

PDC-like cells are pre-DC2 and require KLF4 to control homeostatic CD4 T cells.

Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons to antigen presentation. However, recent studies uncovered unexpected heterogeneity among pDCs. We identified a previously uncharacterized immune subset, referred to as pDC-like cells, that not only resembles pDCs but also shares conventional DC (cDC) features. We show that this subset is a circulating precursor distinct from common DC progenitors, with prominent cDC2 potential. Our findings from human CD2-iCre and CD300c-iCre lineage tracing mouse models suggest that a substantial fraction of cDC2s originates from pDC-like cells, which can therefore be referred to as pre-DC2. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor, by expanding and differentiating into cDC2 that efficiently prime T helper 17 (TH17) cells. Development of pre-DC2 into CX3CR1+ ESAM- cDC2b but not CX3CR1- ESAM+ cDC2a requires the transcription factor KLF4. Last, we show that, under homeostatic conditions, this developmental pathway regulates the immune threshold at barrier sites by controlling the pool of TH17 cells within skin-draining lymph nodes.

Traumatic brain injury alters dendritic cell differentiation and distribution in lymphoid and non-lymphoid organs

BackgroundPathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI.MethodsLong bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 μM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining.ResultsOur studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI.ConclusionsOur data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.

Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation

Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.

Class I PI3K regulatory subunits control differentiation of dendritic cell subsets and regulate Flt3L mediated signal transduction

Dendritic cells (DCs) play pivotal roles in initiating and shaping both innate and adaptive immune responses. The spatiotemporal expression of transcription factor networks and activation of specific signal transduction pathways determine the specification, distribution and differentiation of DC subsets. Even though pioneering studies have established indispensable roles for specific catalytic subunits (p110δ and p110γ) in immune cells, functions of the regulatory subunits, particularly of Class I PI3K, within the hematopoietic system remain incompletely understood. In the study presented here, we deleted the key regulatory subunits—p85α and p85β of the Class IA PI3K in hematopoietic cells and studied its impact on DC differentiation. Our studies identify that a deficiency of p85 causes increased differentiation of conventional DC (cDC) 2 and plasmacytoid DC (pDC) subsets in the spleen. On the other hand, DC numbers in the bone marrow (BM), thymus and lymph nodes were decreased in p85 mutant mice. Analysis of DC-specific progenitors and precursors indicated increased numbers in the BM and spleen of p85 deficient mice. In-vitro differentiation studies demonstrated augmented DC-differentiation capacities of p85 deficient BM cells in the presence of GM-CSF and Flt3L. BM chimera studies established that p85 deficiency affects DC development through cell intrinsic mechanisms. Molecular studies revealed increased proliferation of DCs and common DC progenitors (CDPs) in the absence of p85 and altered signal transduction pathways in p85 mutant DC subsets in response to Flt3L. In essence, data presented here, for the first time, unequivocally establish that the P85α subunit of class IA PI3Ks has an indispensable role in the development and maintenance of DCs.

Ablation of cDC2 lineage specification by mutations within the −165 kb Zeb2 enhancer

Abstract Efforts to determine the unique functions of the two types of classical dendritic cells, cDC1 and cDC2, are hindered by limited understanding of the divergence of the common dendritic cell progenitor (CDP). Some transcription factors act in commitment of already specified progenitors, such as Batf3 which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer, but how other factors control CDP divergence remains unknown. Here, we report the transcriptional mechanism of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis suggested that Nfil3 acts upstream of Id2, Batf3, and Zeb2 in cDC1 development but has not revealed its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and ChIP-seq analysis identified NFIL3 binding in the −165 kb Zeb2 enhancer at sites that also bind CCAAT-enhancer-binding proteins (C/EBPs). Mutation of these NFIL3/C/EBP sites by in vivo CRISPR/Cas9 targeting revealed functional redundancy, with C/EBPs and NFIL3 competing in binding these sites to support or repress Zeb2 expression, respectively. Mutation of these three NFIL3/C/EBP sites eliminated Zeb2 expression in myeloid, but not lymphoid progenitors, producing mice that completely lacked pre-cDC2 specification and mature cDC2 in vivo. These mice failed to make an appropriate TH2 response against H. polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths. Notably, Zeb2 expression is not required to maintain cDC2 identity, but acts only to block cDC1 specification. Thus, the competition between C/EBPs and NFIL3 binding at the −165 kb Zeb2 enhancer determines CDP divergence.

Flt3L, LIF, and IL-10 combination promotes the selective in vitro development of ESAMlow cDC2B from murine bone marrow.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103- XCR1- CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4-/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAMlow cDC2 (cDC2B) development and survival in vitro.

Transition from cMyc to L-Myc during dendritic cell development coordinated by rising levels of IRF8.

During dendritic cell (DC) development, Myc expression in progenitors is replaced by Mycl in mature DCs, but when and how this transition occurs is unknown. We evaluated DC development using reporters for MYC, MYCL, and cell cycle proteins Geminin and CDT1 in wild-type and various mutant mice. For classical type 1 dendritic cells (cDC1s) and plasmacytoid DCs (pDCs), the transition occurred upon their initial specification from common dendritic cell progenitors (CDPs) or common lymphoid progenitors (CLPs), respectively. This transition required high levels of IRF8 and interaction with PU.1, suggesting the use of EICEs within Mycl enhancers. In pDCs, maximal MYCL induction also required the +41kb Irf8 enhancer that controls pDC IRF8 expression. IRF8 also contributed to repression of MYC. While MYC is expressed only in rapidly dividing DC progenitors, MYCL is most highly expressed in DCs that have exited the cell cycle. Thus, IRF8 levels coordinate the Myc-Mycl transition during DC development.

Linking GATA2 Deficiency to Dysregulated Innate Immune Signaling

Cell type-specific transcription factors governing hematopoietic stem and progenitor cell transitions establish networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 mechanisms that control hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent genetic network governing hematopoietic progenitor cell differentiation (Johnson KD. et al., Sci. Adv., 2015). The aberrant network includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators, including Toll-like receptors (TLRs) (Johnson KD. et al., J. Exp. Med., 2020). Mutant embryonic progenitors lose the capacity to balance production of diverse hematopoietic progeny and generate excessive monocytic progeny. As IRF8 is vitally important for monocytic and dendritic cell differentiation (Yanez A. and Goodridge H., Curr. Opin. Hematol., 2016), we asked whether IRF8 is essential, contributory, or inconsequential. Using a double-mutant genetic rescue in vivo system, we demonstrated that reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell-committed progenitors. In -77 -/- E14.5 fetal livers, monocyte progenitors (MPs) increased 2.3-fold (P = 0.006), granulocyte progenitors (GPs) decreased 2.2-fold (P = 0.003) and common dendritic cell progenitors (CDPs) increased 10.2-fold (P = 0.021) relative to wildtype littermates. Ablating Irf8 in -77 mutants (-77 -/-; Irf8-/-) restored MPs and CDPs to wildtype levels and reversed the GP deficiency; further increasing GPs 4.2-fold relative to wildtype (P = 0.0009). Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the embryonic hematopoiesis program. We analyzed the mechanistic and biological implications of IRF8 dysregulation concomitant with ectopic upregulation of other innate immune genes (including Toll-like receptors (TLRs) in GATA2-deficient embryonic progenitors. In principle, such genes might function upstream, downstream, or in parallel with IRF8. Based on TLR upregulation and TLR roles in progenitor mechanisms (Nagai Y. et al., Immunity, 2006; Schuettpelz L. et al., Leukemia, 2014; Caiado F. et al., J. Exp. Med., 2021), we tested whether GATA2 deficiency in embryonic progenitors impacts cellular responsiveness to TLR ligands. Wild type and -77 enhancer-mutant progenitors were treated with increasing concentrations of the TLR1/2 ligand Pam 3CSK 4. The mutant progenitors were hypersensitive to Pam 3CSK 4, which resulted in supra-physiological induction of Tnf expression (2.8-fold at 34 nM, P = 0.004; 3.2-fold at 68 nM, P = 0.0003). Quantitative analyses indicated that hypersensitivity reflected increased Pam 3CSK 4 efficacy, but not potency. GATA2 re-expression in the mutant progenitors attenuated the elevated IRF8 expression and TLR signaling, normalizing Tnf and Ccl3 expression to a level comparable to that of wild type progenitors. In GATA2-rescued mutant progenitors, Tnf and Ccl3 expression decreased 3.9-fold (P = 0.005) and 2.5-fold (P = 0.047), respectively. Thus, GATA2 suppresses TLR signaling in embryonic progenitors. Ongoing studies are elucidating the mechanistic interconnections between IRF8- and TLR-dependent inflammatory networks in GATA2 deficiency during embryonic and adult hematopoiesis in cell populations and single cells, relationships between murine and human mechanisms, and the impact of targeted interventions that modulate these mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Notch Signaling Promotes the Differentiation of Human CLEC9A+ Dendritic Cells from Hematopoietic Stem and Progenitor Cells

Human CLEC9A+ dendritic cells (also known as CD141+ DCs, XCR1+ DCs, and cDC1) are an endogenous DC subset with constitutive ability to traffic to secondary lymphoid tissues, efficiently cross-present cellular antigens, and prime CD4+ and CD8+ antiviral and antitumor T cell responses. As such, CLEC9A+ DCs have been proposed as ideal candidates for adoptive cell immunotherapy, however their rarity in vivo and a lack of knowledge regarding their development have thus far precluded their therapeutic use.We used in vitro differentiation approaches to identify novel microenvironmental cues that regulate the development of CLEC9A+ DCs from human CD34+ hematopoietic stem and progenitor cells (HSPCs). Our findings reveal a previously unknown role for Notch signaling as a key regulatory pathway in CLEC9A+ DC differentiation from HSPCs. Exposure of HSPCs to the Notch ligand DLL1 expressed by MS5 stromal cells, together with the DC-promoting cytokines FLT3L and GM-CSF, markedly enhanced the generation of CLEC9A+ DCs relative to control cultures lacking DLL1 expression. Resulting CLEC9A+ DC frequency was on average 66% of total cells in DLL1 cultures, compared with 6% in control cultures at 20 days (p < 0.0001). Promotion of CLEC9A+ DC development was accompanied by statistically significant decreases in monocyte, CD1c+ DC, and plasmacytoid DC frequencies and cell numbers, with granulocytic differentiation relatively unaffected. Similar changes were seen when DLL1 was substituted for the Notch ligands DLL4 or JAG1, and abrogated by gamma secretase inhibition. Furthermore, promotion of CLEC9A+ DC differentiation was mimicked by transduction of HSPCs with intracellular Notch1, consistent with a cell-intrinsic role for Notch in CLEC9A+ DC development.The effect of Notch signaling on CLEC9A+ DC development was conserved between HSPCs isolated from human cord blood (CB), bone marrow (BM), or G-CSF mobilized peripheral blood (MPB). Furthermore, highly purified BM monocyte/dendritic cell progenitors (MDP) or common dendritic cell progenitors (CDP) cultured in the presence of DLL1 preferentially generated CLEC9A+ DCs with loss of alternative lineage outputs, supporting a mechanism by which Notch signaling skews lineage commitment to the CLEC9A+ DC lineage at the level of multipotent DC progenitors.Notch-induced CLEC9A+ DCs derived from HSPCs were phenotypically and functionally similar to primary CLEC9A+ DCs isolated from the blood. Whole transcriptome profiling by RNAseq of Notch-induced CLEC9A+ DCs confirmed lineage-specific upregulation of key CLEC9A+ DC genes including XCR1, CADM1, BATF3, and IRF8 . Furthermore, global gene expression profiles were similar between Notch-induced CLEC9A+ DCs generated from HSPCs isolated from CB or MPB. Notch-induced CLEC9A+ DCs expressed high levels of CD62L and CCR7, and underwent efficient chemotaxis in response to CCL21 in transwell migration assays, suggesting lymph node-homing capacity. Compared to primary blood CLEC9A+ DCs, Notch-induced CLEC9A+ DCs exhibited higher basal surface expression of T cell costimulatory molecules including CD80, CD83, and CD86, reminiscent of dermal CLEC9A+ DCs, and these were further upregulated in response to the TLR agonists poly(I:C) or R848. Notch-induced CLEC9A+ DCs exhibited potent CD4+ and CD8+ immunostimulatory activity in mixed lymphocyte reactions, and induced antigen-specific CD8+ T cell responses to an HLA-A*0201-restricted NY-ESO-1 epitope through either cross-presentation of cellular antigen acquired from necrotic tumor cells, or endogenous presentation of antigen transduced at the CD34+ HSPC stage.In conclusion, we have identified a novel role for Notch signaling in the differentiation of CLEC9A+ DCs from HSPCs. We propose Notch signaling enforces CLEC9A+ DC lineage commitment in multipotent DC progenitors, and RNAseq studies addressing the molecular basis of Notch-induced CLEC9A+ DC commitment are in progress. Importantly, the identification of Notch as a critical regulatory pathway in human CLEC9A+ DC development allows robust in vitro differentiation of large numbers of highly functional CLEC9A+ DCs. Taken together our findings provide insight into the development of this important DC lineage and permit the preclinical development of next generation cellular vaccine strategies using in vitro derived CLEC9A+ DCs. DisclosuresMontel-Hagen:Kite Pharma: Research Funding. Kohn:Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biogen IDEC: Research Funding; BioMarin Pharmaceutical: Research Funding; Orchard Therapeutics Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Crooks:Kite Pharma: Research Funding.

Commitment to dendritic cells and monocytes.

Dendritic cells (DCs) and monocytes are widely conserved immune cells in vertebrates that arise from hematopoietic stem cells via intermediate progenitors. The progenitors that strictly give rise to DCs or monocytes have been recently identified both in humans and in mice, thereby revealing their differentiation pathways. Advances in analysis technologies have further deepened our understanding of the development of DCs and monocytes from progenitor population-based to individual progenitor cell-based commitment. Since DC-committed progenitors, common DC progenitors (CDPs) and precursor conventional DCs (pre-cDCs) do not differentiate into monocytes, DCs are a distinct lineage from monocytes, although monocytes can acquire DC functions upon activation at tissues where they arrive.

A RUNX-CBFβ-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFβ-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization

There is accumulating evidence suggesting that toll-like receptor (TLR) signals play an important role in the regulation of hematopoietic stem/progenitor cells (HSPCs). TLR7/8 stimulation induces the myeloid differentiation of normal HSPCs and acute myeloid leukemia cells. However, the in vivo effect of TLR7/8 agonists on hematopoiesis is largely unknown. Here, we show that, similar to TLR4 and TLR2, treatment with the TLR7/8 agonist R848 induces an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced repopulating potential and HSPC mobilization. In contrast to chronic TLR4 stimulation, treatment with R848 for 5 days did not induce a significant increase in myeloid-biased HSCs. Treatment with R848 results in a significant increase in classic dendritic cells (DCs) in the bone marrow, but a decrease in common dendritic cell progenitors and pre-DCs. Phenotypic analysis of DCs revealed that R848 treatment is associated with altered expression of certain chemokines, activation markers, and migratory receptors. Together, these data indicate that systemic administration of a TLR7/8 agonist has unique effects on hematopoiesis, including the expansion of DCs in the bone marrow, that might have clinical relevance to augment responses to certain immunotherapies, such as cancer vaccines and immune checkpoint blockade.