IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

Transcriptional Networks Driving Dendritic Cell Differentiation and Function.

Dendritic cells (DCs) are the sentinels of the immune system, sensing a diverse array of pathogens to stimulate a robust and appropriate immune response. To initiate responses to highly disparate challenges, DCs have diversified into multiple phenotypically, anatomically, and functionally distinct cell types. As a result of the application of new single-cell technologies, the full extent of this diversity, as well as the developmental relationships of the DC lineages, is currently undergoing reassessment. Here, we review the cellular and molecular evidence that underpins current models of DC differentiation and functional diversification in the murine and human systems. We discuss these models in the context of the diversity revealed by single-cell studies and propose that understanding DC identity will require defining the regulatory interactions that control gene expression in these cells.

Transcription Factor PU.1 Promotes Conventional Dendritic Cell Identity and Function via Induction of Transcriptional Regulator DC-SCRIPT.

Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.

The transcription factor T-bet is essential for the development of NKp46+ innate lymphocytes via the Notch pathway.

NKp46+ innate lymphoid cells (ILCs) serve important roles in regulating the intestinal microbiota and defense against pathogens. Whether NKp46+ ILCs arise directly from lymphoid tissue-inducer (LTi) cells or represent a separate lineage remains controversial. We report here that the transcription factor T-bet (encoded by Tbx21) was essential for the development of NKp46+ ILCs but not of LTi cells or nuocytes. Deficiency in interleukin 22 (IL-22)-producing NKp46+ ILCs resulted in greater susceptibility of Tbx21-/- mice to intestinal infection. Haploinsufficient T-bet expression resulted in lower expression of the signaling molecule Notch, and Notch signaling was necessary for the transition of LTi cells into NKp46+ ILCs. Furthermore, NKp46+ ILCs differentiated solely from the CD4- LTi population, not the CD4+ LTi population. Our results pinpoint the regulation of Notch signaling by T-bet as a distinct molecular pathway that guides the development of NKp46+ ILCs.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf-/-) or specifically in the gut epithelium. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf-/- mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf-/-mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Type 1 conventional dendritic cells: ontogeny, function, and emerging roles in cancer immunotherapy.

Dendritic cells (DCs) are key immune sentinels that orchestrate protective immune responses against pathogens or cancers. DCs have evolved into multiple phenotypically, anatomically, and functionally distinct cell types. One of these DC types, Type 1 conventional DCs (cDC1s), are uniquely equipped to promote cytotoxic CD8+ T cell differentiation and, therefore, represent a promising target for harnessing antitumor immunity. Indeed, recent studies have highlighted the importance of cDC1s in tumor immunotherapy using immune checkpoint inhibitors. Here, we review the progress in defining the key developmental and functional attributes of cDC1s and the approaches to optimizing the potency of cDC1s for anticancer immunity.

ILC2-derived IL-13 promotes skin cDC2 diversity.

In a new study, researchers have identified a new population of type 2 conventional dendritic cells in the skin that depend on IL-13 and promote Th2 mediated immunity.

ZC3H12C expression in dendritic cells is necessary to prevent lymphadenopathy of skin-draining lymph nodes.

The role of RNA-binding proteins of the CCCH-containing family in regulating proinflammatory cytokine production and inflammation is increasingly recognized. We have identified ZC3H12C (Regnase-3) as a potential post-transcriptional regulator of tumor necrosis factor expression and have investigated its role in vivo by generating Zc3h12c-deficient mice that express green fluorescent protein instead of ZC3H12C. Zc3h12c-deficient mice develop hypertrophic lymph nodes. In the immune system, ZC3H12C expression is mostly restricted to the dendritic cell (DC) populations, and we show that DC-restricted ZC3H12C depletion is sufficient to cause lymphadenopathy. ZC3H12C can regulate Tnf messenger RNA stability via its RNase activity in vitro, and we confirmed the role of Tnf in the development of lymphadenopathy. Finally, we found that loss of ZC3H12C did not impact the outcome of skin inflammation in the imiquimod-induced murine model of psoriasis, despite Zc3h12c being identified as a risk factor for psoriasis susceptibility in several genome-wide association studies. Our data suggest a role for ZC3H12C in DC-driven skin homeostasis.

Plasmacytoid dendritic cells from parent strains of the NZB/W F1 lupus mouse contribute different characteristics to autoimmune propensity.

The NZB/W F1 (F1) mice develop severe disease that is similar to human systemic lupus erythematosus. By contrast, each parent strain, NZB or NZW, has limited autoimmunity, suggesting traits of both strains contribute to pathogenesis. Although many of the contributing genes have been identified, the contributing cellular abnormality associated with each parent strain remains unresolved. Given that plasmacytoid dendritic cells (pDCs) are key to the pathogenesis of lupus, we investigated the properties of pDCs from NZB and NZW mice. We found that NZB mouse had higher numbers of pDCs, with much of the increase being contributed by a more abundant CD8+ pDC subset. This was associated with prolonged survival and stronger proliferation of CD4+ T cells. By contrast, NZW pDCs had heightened capacity to produce interferon-α (IFNα) and IFNλ, and promoted stronger B-cell proliferation upon CpG stimulation. Thus, our data reveal the different functional and numerical characteristics of pDCs from NZW and NZB mouse.

EZH2 function in immune cell development.

The polycomb repressive complex 2 (PRC2) consists of three core components EZH2, SUZ12 and EED. EZH2 catalyzes the methylation of lysine 27 of histone H3, a modification associated with gene silencing. Through gene duplication higher vertebrate genomes also encode a second partially redundant methyltransferase, EZH1. Within the mammalian immune system most research has concentrated on EZH2 which is expressed predominantly in proliferating cells. EZH2 and other PRC2 components are required for hematopoietic stem cell function and lymphocyte development, at least in part by repressing cell cycle inhibitors. At later stages of immune cell differentiation, EZH2 plays essential roles in humoral and cell-mediated adaptive immunity, as well as the maintenance of immune homeostasis. EZH2 is often overactive in cancers, through both gain-of-function mutations and over-expression, an observation that has led to the development and clinical testing of specific EZH2 inhibitors. Such inhibitors may also be of use in inflammatory and autoimmune settings, as EZH2 inhibition dampens the immune response. Here, we will review the current state of understanding of the roles for EZH2, and PRC2 more generally, in the development and function of the immune system.

Establishing and maintaining the Langerhans cell network.

Langerhans cells (LCs) are the unique antigen-presenting cell of the epidermis. LCs have long been depicted in textbooks as the archetypical dendritic cell that alerts the immune system upon pathogen induced skin barrier breakage, however recent findings argue instead for a more tolerogenic function. While the LCs that populate the epidermis in steady-state arise from progenitors that seed the skin during embryogenesis, it is now apparent that a second pathway generating LCs from a bone marrow derived progenitor is active in inflammatory settings. This review emphasizes the determinants underpinning the establishment of the LC network in steady-state and under inflammatory conditions, as well as the transcriptional machinery governing their differentiation. The dual origin of LCs raises important questions about the functional differences between these subsets in balancing the epidermal immune response between immunity and tolerance.

Changes in the sympathetic innervation of the gut in rotenone treated mice as possible early biomarker for Parkinson's disease.

INTRODUCTION: Involvement of the peripheral nervous system (PNS) is relatively common in Parkinson's disease (PD) patients. PNS alterations appear early in the course of the disease and are responsible for some of the non-motor symptoms observed in PD patients. In previous studies, we have shown that environmental toxins can trigger the disease by acting on the enteric nervous system. MATERIAL AND METHODS: Here, we analyzed the effect of mitochondrial Complex I inhibition on sympathetic neuritis in vivo and sympathetic neurons in vitro. Combining in vivo imaging and protein expression profiling. RESULTS: we found that rotenone, a widely used mitochondrial Complex I inhibitor decreases the density of sympathetic neurites innervating the gut in vivo, while in vitro, it induces the redistribution of intracellular alpha-synuclein and neurite degeneration. Interestingly, sympathetic neurons are much more resistant to rotenone exposure than mesencephalic dopaminergic neurons. CONCLUSION: Altogether, these results suggest that enteric sympathetic denervation could be an initial pre-motor alteration in PD progression that could be used as an early biomarker of the disease.

Genome-wide DNA methylation analysis identifies hypomethylated genes regulated by FOXP3 in human regulatory T cells.

Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4(+) T cells (Naive). We detected 2315 differentially methylated cytosine-guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

Mis-expression of GATA6 re-programs cell fate during early hematopoiesis.

The traditional view of hematopoiesis is that myeloid cells derive from a common myeloid progenitor (CMP), whereas all lymphoid cell populations, including B, T, and natural killer (NK) cells and possibly plasmacytoid dendritic cells (pDCs), arise from a common lymphoid progenitor (CLP). In Max41 transgenic mice, nearly all B cells seem to be diverted into the granulocyte lineage. Here, we show that these mice have an excess of myeloid progenitors, but their CLP compartment is ablated, and they have few pDCs. Nevertheless, T cell and NK cell development proceeds relatively normally. These hematopoietic abnormalities result from aberrant expression of Gata6 due to serendipitous insertion of the transgene enhancer (Eµ) in its proximity. Gata6 mis-expression in Max41 transgenic progenitors promoted the gene-regulatory networks that drive myelopoiesis through increasing expression of key transcription factors, including PU.1 and C/EBPa. Thus, mis-expression of a single key regulator like GATA6 can dramatically re-program multiple aspects of hematopoiesis.

Sphingosine 1-phosphate-induced motility and endocytosis of dendritic cells is regulated by SWAP-70 through RhoA.

The phospholipid mediator sphingosine 1-phosphate (S1P) enhances motility and endocytosis of mature dendritic cells (DCs). We show that in vitro migration of Swap-70(-/-) bone marrow-derived DCs (BMDCs) in response to S1P and S1P-induced upregulation of endocytosis are significantly reduced. S1P-stimulated movement of Swap-70(-/-) BMDCs, specifically retraction of their trailing edge, in a collagen three-dimensional environment is impaired. These in vitro observations correlate with delayed entry into lymphatic vessels and migration to lymph nodes of skin DCs in Swap-70(-/-) mice. Expression of S1P receptors (S1P(1-3)) by wild-type and Swap-70(-/-) BMDCs is similar, but Swap-70(-/-) BMDCs fail to activate RhoA and to localize Rac1 and RhoA into areas of actin polymerization after S1P stimulus. The Rho-activating G protein Gα(i) interacts with SWAP-70, which also supports the localization of Gα(13) to membrane rafts in BMDCs. LPS-matured Swap-70(-/-) BMDCs contain significantly more active RhoA than wild-type DCs. Preinhibition of Rho activation restored migration to S1P, S1P-induced upregulation of endocytosis in mature Swap-70(-/-) BMDCs, and localization of Gα(13) to membrane rafts. These data demonstrate SWAP-70 as a novel regulator of S1P signaling necessary for DC motility and endocytosis.

Transcriptional regulation of dendritic cell development and function.

Dendritic cells (DCs) are sentinel immune cells that form a critical bridge linking the innate and adaptive immune systems. Extensive research addressing the cellular origin and heterogeneity of the DC network has revealed the essential role played by the spatiotemporal activity of key transcription factors. In response to environmental signals DC mature but it is only following the sensing of environmental signals that DC can induce an antigen specific T cell response. Thus, whilst the coordinate action of transcription factors governs DC differentiation, sensing of environmental signals by DC is instrumental in shaping their functional properties. In this review, we provide an overview that focuses on recent advances in understanding the transcriptional networks that regulate the development of the reported DC subsets, shedding light on the function of different DC subsets. Specifically, we discuss the emerging knowledge on the heterogeneity of cDC2s, the ontogeny of pDCs, and the newly described DC subset, DC3. Additionally, we examine critical transcription factors such as IRF8, PU.1, and E2-2 and their regulatory mechanisms and downstream targets. We highlight the complex interplay between these transcription factors, which shape the DC transcriptome and influence their function in response to environmental stimuli. The information presented in this review provides essential insights into the regulation of DC development and function, which might have implications for developing novel therapeutic strategies for immune-related diseases.