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Epithelial tuft cells are shown to be the source of intestinal interleukin (IL)-25 that is required for activation of type 2 innate lymphoid cells (ILC2s), ILC2-regulated tuft and goblet cell expansion, and control of parasite infection. Epithelial tuft cells in type 2 immunity The 'weep-and-sweep' response to parasitic helminths and allergens, in which parasites are ejected by increased propulsive activity of the gut combined with fluid and mucus secretion, is a manifestation of type 2 (or allergic) immunity involving the activation of group 2 innate lymphoid cells (ILC2s). The epithelium in the small intestine consists of five or more distinct cellular lineages, including tuft cells, whose functions remain unclear. Two papers in this issue of Nature demonstrate a role for tuft cells in the response to parasites. Richard Locksley and colleagues show that tuft cells are the source of the interleukin 25 (IL-25) that is required for activation of ILC2s, ILC2-regulated tuft and goblet cell expansion, and control of parasite infection. Philippe Jay and colleagues show that tuft cells secrete IL-25 via an IL-13/IL-4R -dependent feedback loop. Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells 1 and smooth muscle hypercontractility 2 . This response, known as ‘weep and sweep’, requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (T H 2 cells) 3 . Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages 12 , including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells.

Regulatory T cells expressing the transcription factor T-bet selectively suppress T H 1 and CD8 T cells, but not T H 2 or T H 17 activation and associated autoimmunity. Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (T H 1), T H 2, and T H 17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively 1 . Regulatory T (T reg ) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2 , 3 ). Paradoxically, some activated T reg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide T reg cells with enhanced suppressive capacity 4 , 5 , 6 . Whether expression of these factors in T reg cells—as in effector T cells—is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the T H 1-associated transcription factor T-bet in mouse T reg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing T reg cells—but not of T-bet expression in T reg cells—resulted in severe T H 1 autoimmunity. Conversely, following depletion of T-bet − T reg cells, the remaining T-bet + cells specifically inhibited T H 1 and CD8 T cell activation consistent with their co-localization with T-bet + effector T cells. These results suggest that T-bet + T reg cells have an essential immunosuppressive function and indicate that T reg cell functional heterogeneity is a critical feature of immunological tolerance.

Epithelial tuft cells secretion of IL-25 is shown to regulate type 2 epithelial responses to helminth parasite infection via an IL-13/IL-4Rα-dependent feedback loop. Epithelial tuft cells in type 2 immunity The 'weep-and-sweep' response to parasitic helminths and allergens, in which parasites are ejected by increased propulsive activity of the gut combined with fluid and mucus secretion, is a manifestation of type 2 (or allergic) immunity involving the activation of group 2 innate lymphoid cells (ILC2s). The epithelium in the small intestine consists of five or more distinct cellular lineages, including tuft cells, whose functions remain unclear. Two papers in this issue of Nature demonstrate a role for tuft cells in the response to parasites. Richard Locksley and colleagues show that tuft cells are the source of the interleukin 25 (IL-25) that is required for activation of ILC2s, ILC2-regulated tuft and goblet cell expansion, and control of parasite infection. Philippe Jay and colleagues show that tuft cells secrete IL-25 via an IL-13/IL-4R -dependent feedback loop. Helminth parasitic infections are a major global health and social burden 1 . The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity 2 . Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion 3 , 4 . However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium 5 , are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature 6 and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3 −/− mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

The divergence of the common dendritic cell progenitor 1 – 3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages 4 , 5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors—such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer 4 , 6 —but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis 7 suggested that Nfil3 acts upstream of Id2 , Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the –165 kb Zeb2 enhancer 8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPβ. In vivo mutational analysis using CRISPR–Cas9 targeting showed that these NFIL3–C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3–C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (T H 2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting T H 2 responses to helminths 9 – 11 . Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the –165 kb Zeb2 enhancer. The transcription factor NFIL3 acts antagonistically to C/EBP proteins by binding the Zeb2 enhancer to prevent Zeb2 expression and the development of the conventional type 2 dendritic cell lineage.

Intergenic long noncoding RNAs (lincRNAs) regulate gene expression in various tissues. Zhao and colleagues identify 1,524 lincRNA clusters in thymocytes and mature T cell subsets and reveal dynamic and cell-specific patterns of lincRNA expression during T cell differentiation. Although intergenic long noncoding RNAs (lincRNAs) have been linked to gene regulation in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identified 1,524 lincRNA clusters in 42 T cell samples, from early T cell progenitors to terminally differentiated helper T cell subsets. Our analysis revealed highly dynamic and cell-specific expression patterns for lincRNAs during T cell differentiation. These lincRNAs were located in genomic regions enriched for genes that encode proteins with immunoregulatory functions. Many were bound and regulated by the key transcription factors T-bet, GATA-3, STAT4 and STAT6. We found that the lincRNA LincR- Ccr2 -5′AS, together with GATA-3, was an essential component of a regulatory circuit in gene expression specific to the T H 2 subset of helper T cells and was important for the migration of T H 2 cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.

The balance between T helper type 1 (T H 1) cells and other T H cells is critical for antiviral and anti-tumour responses 1 – 3 , but how this balance is achieved remains poorly understood. Here we dissected the dynamic regulation of T H 1 cell differentiation during in vitro polarization, and during in vivo differentiation after acute viral infection. We identified regulators modulating T helper cell differentiation using a unique T H 1–T H 2 cell dichotomous culture system and systematically validated their regulatory functions through multiple in vitro and in vivo CRISPR screens. We found that RAMP3, a component of the receptor for the neuropeptide CGRP (calcitonin gene-related peptide), has a cell-intrinsic role in T H 1 cell fate determination. Extracellular CGRP signalling through the receptor RAMP3–CALCRL restricted the differentiation of T H 2 cells, but promoted T H 1 cell differentiation through the activation of downstream cAMP response element-binding protein (CREB) and activating transcription factor 3 (ATF3). ATF3 promoted T H 1 cell differentiation by inducing the expression of Stat1 , a key regulator of T H 1 cell differentiation. After viral infection, an interaction between CGRP produced by neurons and RAMP3 expressed on T cells enhanced the anti-viral IFNγ-producing T H 1 and CD8 + T cell response, and timely control of acute viral infection. Our research identifies a neuroimmune circuit in which neurons participate in T cell fate determination by producing the neuropeptide CGRP during acute viral infection, which acts on RAMP3-expressing T cells to induce an effective anti-viral T H 1 cell response. RAMP3, a component of the receptor for the neuropeptide CGRP, has a cell-intrinsic role in T helper type 1 cell fate determination.

Key Points Mast cells and basophils are phenotypically and functionally related cell types that promote protective immunity to helminths, but that can also cause pathology during allergic inflammation. Mast cells and basophils might have evolved from a common precursor cell, such as the mast cell/basophil-like (MCBL) cell or the test cell, which were identified in the urochordate Styela plicata . A basophil/mast cell progenitor (BMCP) cell population was isolated from the spleen of adult mice, and the timed expression of the transcription factors GATA-binding protein 2 and CCAAT/enhancer-binding protein-α in these progenitor cells might determine whether they eventually mature into basophils or mast cells. New mouse models have been developed in which mast cells or basophils can be constitutively or conditionally depleted. These models allow more specific targeting of mast cells and basophils, compared with the Kit -mutant mouse strains and depleting antibody strategies which were previously used to assess mast cell and basophil functions in vivo . Therefore, these new mouse models will help to further clarify the true biological roles of basophils and mast cells. Although mast cells and basophils can express interleukin-4, CD40 ligand and low levels of MHC class II under certain conditions, they seem to have no major role in driving the induction of adaptive type 2 immune responses. Mast cells and basophils cooperate to provide protection during secondary infestation with ticks. In addition, both cell types contribute, to various degrees, to protective immunity during gastrointestinal helminth infections in mice. IgE-mediated anaphylaxis is strictly mast cell-dependent, whereas IgG1-mediated anaphylaxis in mice results from the activation of monocytes, neutrophils or basophils. Basophils are recruited to the skin in a mouse model of atopic dermatitis and they induce IgE-mediated chronic allergic inflammation of the skin in the absence of mast cells. Mast cells and basophils are associated with protective immunity to helminths, but can also drive pathological immune responses in asthma and allergy. This Review covers the recent advances that have improved our understanding of the origins of these cells and of their biological functions in both health and disease. Mast cells and basophils are potent effector cells of the innate immune system, and they have both beneficial and detrimental functions for the host. They are mainly implicated in pro-inflammatory responses to allergens but can also contribute to protection against pathogens. Although both cell types were identified more than 130 years ago by Paul Ehrlich, their in vivo functions remain poorly understood. The precursor cell populations that give rise to mast cells and basophils have recently been characterized and isolated. Furthermore, new genetically modified mouse strains have been developed, which enable more specific targeting of mast cells and basophils. Such advances offer new opportunities to uncover the true in vivo activities of these cells and to revisit their previously proposed effector functions.

Hidden cell sub-populations are detected by accounting for confounding variation inthe analysis of single-cell RNA-seq data. Recent technical developments have enabled the transcriptomes of hundreds of cells to be assayed in an unbiased manner, opening up the possibility that new subpopulations of cells can be found. However, the effects of potential confounding factors, such as the cell cycle, on the heterogeneity of gene expression and therefore on the ability to robustly identify subpopulations remain unclear. We present and validate a computational approach that uses latent variable models to account for such hidden factors. We show that our single-cell latent variable model (scLVM) allows the identification of otherwise undetectable subpopulations of cells that correspond to different stages during the differentiation of naive T cells into T helper 2 cells. Our approach can be used not only to identify cellular subpopulations but also to tease apart different sources of gene expression heterogeneity in single-cell transcriptomes.

CD4(+) T helper 2 (T(H)2) cells secrete interleukin (IL)4, IL5 and IL13, and are required for immunity to gastrointestinal helminth infections. However, T(H)2 cells also promote chronic inflammation associated with asthma and allergic disorders. The non-haematopoietic-cell-derived cytokines thymic stromal lymphopoietin, IL33 and IL25 (also known as IL17E) have been implicated in inducing T(H)2 cell-dependent inflammation at mucosal sites, but how these cytokines influence innate immune responses remains poorly defined. Here we show that IL25, a member of the IL17 cytokine family, promotes the accumulation of a lineage-negative (Lin(-)) multipotent progenitor (MPP) cell population in the gut-associated lymphoid tissue that promotes T(H)2 cytokine responses. The IL25-elicited cell population, termed MPP(type2) cells, was defined by the expression of Sca-1 (also known as Ly6a) and intermediate expression of c-Kit (c-Kit(int)), and exhibited multipotent capacity, giving rise to cells of monocyte/macrophage and granulocyte lineages both in vitro and in vivo. Progeny of MPP(type2) cells were competent antigen presenting cells, and adoptive transfer of MPP(type2) cells could promote T(H)2 cytokine responses and confer protective immunity to helminth infection in normally susceptible Il25(-/-) mice. The ability of IL25 to induce the emergence of an MPP(type2) cell population identifies a link between the IL17 cytokine family and extramedullary haematopoiesis, and suggests a previously unrecognized innate immune pathway that promotes T(H)2 cytokine responses at mucosal sites.