Lrig1- and Wnt-dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis.

The barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment-producing melanocytes and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented 'scale' and hypopigmented 'interscale' epidermis. Why and how mature melanocytes accumulate in scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte and melanin independent and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovers cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

Guidelines for mouse and human DC generation.

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.

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

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 nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. 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 coauthors, making it an essential resource for basic and clinical DC immunologists.

Distinct Murine Mucosal Langerhans Cell Subsets Develop from Pre-dendritic Cells and Monocytes.

Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. We found that, in contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103(+)CD11b(lo) (CD103(+)) and CD11b(+)CD103(-) (CD11b(+)) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103(+) LCs originate from pre-DCs, whereas CD11b(+) LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, the transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with the epithelial position, morphology, and expression of the LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DC, and monocytic) in steady state.

CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells.

HoxB8 multipotent progenitors (MPP) are obtained by expression of the estrogen receptor hormone binding domain (ERHBD) HoxB8 fusion gene in mouse BM cells. HoxB8 MPP generate (i) the full complement of DC subsets (cDC1, cDC2, and pDC) in vitro and in vivo and (ii) allow CRISPR/Cas9-mediated gene editing, for example, generating homozygous deletions in cis-acting DNA elements at high precision, and (iii) efficient gene repression by dCas9-KRAB for studying gene regulation in DC differentiation.

Two distinct types of Langerhans cells populate the skin during steady state and inflammation.

Langerhans cells (LCs), the dendritic cells (DCs) in skin epidermis, possess an exceptional life cycle and developmental origin. Here we identified two types of LCs, short-term and long-term LCs, which transiently or stably reconstitute the LC compartment, respectively. Short-term LCs developed from Gr-1(hi) monocytes under inflammatory conditions and occurred independently of the transcription factor Id2. Long-term LCs arose from bone marrow in steady state and were critically dependent on Id2. Surface marker and gene expression analysis positioned short-term LCs close to Gr-1(hi) monocytes, which is indicative of their monocytic origin. We also show that LC reconstitution after UV light exposure occurs in two waves: an initial fast and transient wave of Gr-1(hi) monocyte-derived short-term LCs is followed by a second wave of steady-state precursor-derived long-term LCs. Our data demonstrate the presence of two types of LCs that develop through different pathways in inflammation and steady state.

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells.

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross-presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8-/- iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8-/- DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898-908.

Differential peak calling of ChIP-seq signals with replicates with THOR.

The study of changes in protein-DNA interactions measured by ChIP-seq on dynamic systems, such as cell differentiation, response to treatments or the comparison of healthy and diseased individuals, is still an open challenge. There are few computational methods comparing changes in ChIP-seq signals with replicates. Moreover, none of these previous approaches addresses ChIP-seq specific experimental artefacts arising from studies with biological replicates. We propose THOR, a Hidden Markov Model based approach, to detect differential peaks between pairs of biological conditions with replicates. THOR provides all pre- and post-processing steps required in ChIP-seq analyses. Moreover, we propose a novel normalization approach based on housekeeping genes to deal with cases where replicates have distinct signal-to-noise ratios. To evaluate differential peak calling methods, we delineate a methodology using both biological and simulated data. This includes an evaluation procedure that associates differential peaks with changes in gene expression as well as histone modifications close to these peaks. We evaluate THOR and seven competing methods on data sets with distinct characteristics from in vitro studies with technical replicates to clinical studies of cancer patients. Our evaluation analysis comprises of 13 comparisons between pairs of biological conditions. We show that THOR performs best in all scenarios.

The clash of Langerhans cell homeostasis in skin: Should I stay or should I go?

Langerhans cells (LC), the skin epidermal contingent of dendritic cells (DC), possess an exceptional life cycle and developmental origin. LC, like all mature blood cells, develop from haematopoietic stem cells (HSC) through successive steps of lineage commitment and differentiation. However, LC development is different to that of other DC subsets and not yet fully understood. Haematopoietic cell fate decisions are instructed by specific growth factors and cytokines produced in specialized microenvironments or niches. Upon ligand binding the cognate surface receptors on HSC and further restricted progenitor cells regulate the signalling pathways that eventually leads to the execution of lineage-determining genetic programs. In this review we focus on a specific set of surface receptor kinases that have been identified as critical regulators of LC development using genetically modified mice. Recent studies suggest for some of these kinases to impact on LC/LC progenitor interaction with the local niche by regulating adhesion and/or migration. During embryonic development, in wound healing and aberrantly in tumour invasion the same kinase receptors control a genetic program known as epithelial-to-mesenchymal-transition (EMT). We will discuss how EMT and its reverse program of mesenchymal-to-epithelial-transition (MET) can serve as universal concepts operating also in LC development.

Epigenetic program and transcription factor circuitry of dendritic cell development.

Dendritic cells (DC) are professional antigen presenting cells that develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Multipotent progenitors (MPP) are committed to DC restricted common DC progenitors (CDP), which differentiate into specific DC subsets, classical DC (cDC) and plasmacytoid DC (pDC). To determine epigenetic states and regulatory circuitries during DC differentiation, we measured consecutive changes of genome-wide gene expression, histone modification and transcription factor occupancy during the sequel MPP-CDP-cDC/pDC. Specific histone marks in CDP reveal a DC-primed epigenetic signature, which is maintained and reinforced during DC differentiation. Epigenetic marks and transcription factor PU.1 occupancy increasingly coincide upon DC differentiation. By integrating PU.1 occupancy and gene expression we devised a transcription factor regulatory circuitry for DC commitment and subset specification. The circuitry provides the transcription factor hierarchy that drives the sequel MPP-CDP-cDC/pDC, including Irf4, Irf8, Tcf4, Spib and Stat factors. The circuitry also includes feedback loops inferred for individual or multiple factors, which stabilize distinct stages of DC development and DC subsets. In summary, here we describe the basic regulatory circuitry of transcription factors that drives DC development.

Dendritic cell development requires histone deacetylase activity.

DCs develop from multipotent progenitors (MPPs), which commit into DC-restricted common dendritic cell progenitors (CDPs). CDPs further differentiate into classical DCs (cDCs) and plasmacytoid DCs (pDCs). Here, we studied the impact of histone acetylation on DC development in C57BL/6 mice by interfering with histone acetylation and deacetylation, employing histone deacetylase (HDAC) inhibitors. We observed that commitment of MPPs into CDPs was attenuated by HDAC inhibition and that pDC development was specifically blocked. Gene expression profiling revealed that HDAC inhibition prevents establishment of a DC-specific gene expression repertoire. Importantly, protein levels of the core DC transcription factor PU.1 were reduced in HDAC inhibitor-treated cells and consequently PU.1 recruitment at PU.1 target genes Fms-like tyrosine kinase 3 (Flt3), interferon regulatory factor 8 (IRF8), and PU.1 itself was impaired. Thus, our results demonstrate that attenuation of PU.1 expression by HDAC inhibition causes reduced expression of key DC regulators, which results in attenuation of DC development. We propose that chromatin modifiers, such as HDACs, are required for establishing a DC gene network, where Flt3/STAT3 signaling drives PU.1 and IRF8 expression and DC development. Taken together, our study identifies HDACs as critical regulators of DC lineage commitment and development.

Detecting differential peaks in ChIP-seq signals with ODIN.

MOTIVATION: Detection of changes in deoxyribonucleic acid (DNA)-protein interactions from ChIP-seq data is a crucial step in unraveling the regulatory networks behind biological processes. The simplest variation of this problem is the differential peak calling (DPC) problem. Here, one has to find genomic regions with ChIP-seq signal changes between two cellular conditions in the interaction of a protein with DNA. The great majority of peak calling methods can only analyze one ChIP-seq signal at a time and are unable to perform DPC. Recently, a few approaches based on the combination of these peak callers with statistical tests for detecting differential digital expression have been proposed. However, these methods fail to detect detailed changes of protein-DNA interactions. RESULTS: We propose an One-stage DIffereNtial peak caller (ODIN); an Hidden Markov Model-based approach to detect and analyze differential peaks (DPs) in pairs of ChIP-seq data. ODIN performs genomic signal processing, peak calling and p-value calculation in an integrated framework. We also propose an evaluation methodology to compare ODIN with competing methods. The evaluation method is based on the association of DPs with expression changes in the same cellular conditions. Our empirical study based on several ChIP-seq experiments from transcription factors, histone modifications and simulated data shows that ODIN outperforms considered competing methods in most scenarios.

Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation.

Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.

TGF-beta1 accelerates dendritic cell differentiation from common dendritic cell progenitors and directs subset specification toward conventional dendritic cells.

Dendritic cells (DCs) in lymphoid tissue comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that develop from common DC progenitors (CDPs). CDPs are Flt3(+)c-kit(int)M-CSFR(+) and reside in bone marrow. In this study, we describe a two-step culture system that recapitulates DC development from c-kit(hi)Flt3(-/lo) multipotent progenitors (MPPs) into CDPs and further into cDC and pDC subsets. MPPs and CDPs are amplified in vitro with Flt3 ligand, stem cell factor, hyper-IL-6, and insulin-like growth factor-1. The four-factor mixture readily induces self-renewal of MPPs and their progression into CDPs and has no self-renewal activity on CDPs. The amplified CDPs respond to all known DC poietins and generate all lymphoid tissue DCs in vivo and in vitro. Additionally, in vitro CDPs recapitulate the cell surface marker and gene expression profile of in vivo CDPs and possess a DC-primed transcription profile. TGF-ß1 impacts on CDPs and directs their differentiation toward cDCs. Genome-wide gene expression profiling of TGF-ß1-induced genes identified instructive transcription factors for cDC subset specification, such as IFN regulatory factor-4 and RelB. TGF-ß1 also induced the transcription factor inhibitor of differentiation/DNA binding 2 that suppresses pDC development. Thus, TGF-ß1 directs CDP differentiation into cDCs by inducing both cDC instructive factors and pDC inhibitory factors.

Human DC3 Antigen Presenting Dendritic Cells From Induced Pluripotent Stem Cells.

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias toward erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.

Epigenetic aspects of DC development and differentiation.

In this review we introduce the basic principles of epigenetic gene regulation and discuss them in the context of dendritic cell (DC) development and differentiation. Epigenetic mechanisms control the accessibility of chromatin for DNA binding proteins and thus they control gene expression. These mechanisms comprise chemical modifications of DNA and histones, chromatin remodeling and chromatin conformation. The variety of epigenetic mechanisms allow high-end fine tuning and flexibility of gene expression, a prerequisite in the process of DC lineage development.

Distinct Distribution of RTN1A in Immune Cells in Mouse Skin and Lymphoid Organs.

The endoplasmic reticulum-associated protein reticulon 1A (RTN1A) is primarily expressed in neuronal tissues but was recently identified also specifically in cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LCs) and dermal DCs in human skin. In this study, we found that in mice major histocompatibility complex class II (MHCII)+CD207+ LCs but not dermal DCs express RTN1A. Further, RTN1A expression was identified in CD45+MHCII+CD207+ cells of the lymph node and spleen but not in the thymus. Of note, RTN1A was expressed in CD207 low LCs in adult skin as well as emigrated LCs and DCs in lymph nodes and marginally in CD207 hi cells. Ontogeny studies revealed that RTN1A expression occurred before the appearance of the LC markers MHCII and CD207 in LC precursors, while dermal DC and T cell precursors remained negative during skin development. Analogous to the expression of RTN1A in neural tissue, we identified expression of RTN1A in skin nerves. Immunostaining revealed co-localization of RTN1A with nerve neurofilaments only in fetal but not in newborn or adult dermis. Our findings suggest that RTN1A might be involved in the LC differentiation process given its early expression in LC precursors and stable expression onward. Further analysis of the RTN1A expression pattern will enable the elucidation of the functional roles of RTN1A in both the immune and the nervous system of the skin.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.