Fetal lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch (PP) organogenesis, but where these specialized group 3 innate lymphoid cells (ILC3s) develop remains unclear. Here, we identify extrahepatic arginase-1(+) Id2(+) fetal ILC precursors that express a transitional developmental phenotype (ftILCPs) and differentiate into ILC1s, ILC2s and ILC3s in vitro. These cells populate the intestine by embryonic day (E) 13.5 and, before PP organogenesis (E14.5-15), are broadly dispersed in the proximal gut, correlating with regions where PPs first develop. At E16.5, after PP development begins, ftILCPs accumulate at PP anlagen in a lymphotoxin-α-dependent manner. Thus, ftILCPs reside in the intestine during PP development, where they aggregate at PP anlagen after stromal cell activation and become a localized source of ILC populations.

Group 2 innate lymphoid cells (ILC2s) are distributed systemically and produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Transcriptional profiling of ILC2s from different tissues, however, grouped ILC2s according to their tissue of origin, even in the setting of combined IL-25-, IL-33-receptor-, and TSLP-receptor-deficiency. Single-cell profiling confirmed a tissue-organizing transcriptome and identified ILC2 subsets expressing distinct activating receptors, including the major subset of skin ILC2s, which were activated preferentially by IL-18. Tissue ILC2 subsets were unaltered in number and expression in germ-free mice, suggesting that endogenous, tissue-derived signals drive the maturation of ILC2 subsets by controlling expression of distinct patterns of activating receptors, thus anticipating tissue-specific perturbations occurring later in life.

Eosinophils are specialized myeloid cells associated with allergy and helminth infections. Blood eosinophils demonstrate circadian cycling, as described over 80 years ago, and are abundant in the healthy gastrointestinal tract. Although a cytokine, interleukin (IL)-5, and chemokines such as eotaxins mediate eosinophil development and survival, and tissue recruitment, respectively, the processes underlying the basal regulation of these signals remain unknown. Here we show that serum IL-5 levels are maintained by long-lived type 2 innate lymphoid cells (ILC2) resident in peripheral tissues. ILC2 cells secrete IL-5 constitutively and are induced to co-express IL-13 during type 2 inflammation, resulting in localized eotaxin production and eosinophil accumulation. In the small intestine where eosinophils and eotaxin are constitutive, ILC2 cells co-express IL-5 and IL-13; this co-expression is enhanced after caloric intake. The circadian synchronizer vasoactive intestinal peptide also stimulates ILC2 cells through the VPAC2 receptor to release IL-5, linking eosinophil levels with metabolic cycling. Tissue ILC2 cells regulate basal eosinophilopoiesis and tissue eosinophil accumulation through constitutive and stimulated cytokine expression, and this dissociated regulation can be tuned by nutrient intake and central circadian rhythms.

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

Chitin, a polysaccharide constituent of many allergens and parasites, initiates innate type 2 lung inflammation through incompletely defined pathways. We show that inhaled chitin induced expression of three epithelial cytokines, interleukin-25 (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP), which nonredundantly activated resident innate lymphoid type 2 cells (ILC2s) to express IL-5 and IL-13 necessary for accumulation of eosinophils and alternatively activated macrophages (AAMs). In the absence of all three epithelial cytokines, ILC2s normally populated the lung but failed to increase IL-5 and IL-13. Although eosinophils and AAMs were attenuated, neutrophil influx remained normal without these epithelial cytokines. Genetic ablation of ILC2s, however, enhanced IL-1β, TNFα, and IL-23 expression, increased activation of IL-17A-producing γδ T cells, and prolonged neutrophil influx. Thus, chitin elicited patterns of innate cytokines that targeted distinct populations of resident lymphoid cells, revealing divergent but interacting pathways underlying the tissue accumulation of specific types of inflammatory myeloid cells.

Vertebrate immunity has evolved a modular architecture in response to perturbations. Allergic inflammation represents such a module, with signature features of antigen-specific IgE and tissue eosinophilia, although the cellular and molecular circuitry coupling these responses remains unclear. Here, we use genetic and imaging approaches in models of IgE-dependent eosinophilic dermatitis to demonstrate a requisite role for basophils. After antigenic inflammation, basophils initiate transmigration like other granulocytes but, upon activation via their high-affinity IgE receptor, alter their migratory kinetics to persist at the endothelium. Prolonged basophil-endothelial interactions, in part dependent on activation of focal adhesion kinases, promote delivery of basophil-derived IL-4 to the endothelium and subsequent induction of endothelial vascular cell adhesion molecule-1 (VCAM-1), which is required for eosinophil accumulation. Thus, basophils are gatekeepers that link adaptive immunity with innate effector programs by altering access to tissue sites by activation-induced interactions with the endothelium.

Interleukin 4 (IL-4) and IL-13 are critical for responses to parasitic helminthes. We used genetically engineered reporter mice to assess the temporal and spatial production of these cytokines in vivo. In lymph nodes, IL-4, but not IL-13, was made by follicular helper T cells (T(FH) cells). In contrast, tissue type 2 helper T cells (T(H)2 cells) produced both cytokines. There was also divergent production of IL-4 and IL-13 among cells of the innate immune system, whereby basophils produced IL-4, whereas innate helper type 2 cells (Ih2 cells) produced IL-13. IL-13 production by T(H)2 and Ih2 cells was dependent on the transcription factor GATA-3, which was present in large amounts in these cells, and in contrast to the small amount of GATA-3 in T(FH) cells and basophils. The distinct localization and cellular expression of IL-4 and IL-13 explains their unique roles during allergic immunity.

Conditional knockout mice are a useful tool to study the function of gene products in a tissue-specific or inducible manner. Classical approaches to generate targeting vectors for conditional alleles are often limited by the availability of suitable restriction sites. Furthermore, plasmid-based targeting vectors can only cover a few kB of DNA which precludes the generation of targeting vectors where the two loxP sites are placed far apart. These limitations have been overcome in the recent past by using homologous recombination of bacterial artificial chromosomes (BACs) in Escherichia coli to produce large targeting vector containing two different loxP-flanked selection cassettes so that a single targeting event is sufficient to introduce loxP-sites a great distances into the mouse genome. However, the final targeted allele should be free of selection cassettes and screening for correct removal of selection cassettes can be a laborious task. Therefore, we developed a new strategy to rapidly identify ES cells containing the desired allele.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

Zap70 plays a critical role in normal T cell development and T cell function. However, little is known about how perturbation of allosteric autoinhibitory mechanisms in Zap70 impacts T cell biology. Here, we analyze mice with a hypermorphic Zap70 mutation, W131A, which destabilizes the autoinhibitory conformation of Zap70, rendering the kinase in a semiactive state. W131A mutant mice with wild-type T cell receptor (TCR) repertoires exhibited relatively normal T cell development. However, crossing the W131A mutant mice to OTII TCR transgenic mice resulted in increased negative selection of OTII+ thymocytes and in increased thymic and peripheral T regulatory cells. Strikingly, increased basal TCR signaling was associated with a marked increase in inhibitory receptor expression and with T cells that were relatively refractory to TCR stimulation. PD-1 inhibitory receptor blockade partially reversed T cell unresponsiveness. Collectively, disruption of normal Zap70 autoinhibition engaged negative feedback mechanisms by which negative selection and inhibitory receptors restrain TCR signaling to enforce both central and peripheral tolerance.

Interleukin (IL)-17A plays an important role in host defense against a variety of pathogens and may also contribute to the pathogenesis of autoimmune diseases. However, precise identification and quantification of the cells that produce this cytokine in vivo have not been performed. We generated novel IL-17A reporter mice to investigate expression of IL-17A during Klebsiella pneumoniae infection and during experimental autoimmune encephalomyelitis, conditions previously demonstrated to potently induce IL-17A production. In both settings, the majority of IL-17A was produced by non-CD4(+) T cells, particularly γδ T cells, but also invariant NKT cells and other CD4(-)CD3ε(+) cells. As measured in dual-reporter mice, IFN-γ-producing Th1 cells greatly outnumbered IL-17A-producing Th17 cells throughout both challenges. Production of IL-17A by cells from unchallenged mice or by non-T cells under any condition was not evident. Administration of IL-1β and/or IL-23 elicited rapid production of IL-17A by γδ T cells, invariant NKT cells and other CD4(-)CD3ε(+) cells in vivo, demonstrating that these cells are poised for rapid cytokine production and likely comprise the major sources of this cytokine during acute immunologic challenges.

Eosinophils in visceral adipose tissue (VAT) have been implicated in metabolic homeostasis and the maintenance of alternatively activated macrophages (AAMs). The absence of eosinophils can lead to adiposity and systemic insulin resistance in experimental animals, but what maintains eosinophils in adipose tissue is unknown. We show that interleukin-5 (IL-5) deficiency profoundly impairs VAT eosinophil accumulation and results in increased adiposity and insulin resistance when animals are placed on a high-fat diet. Innate lymphoid type 2 cells (ILC2s) are resident in VAT and are the major source of IL-5 and IL-13, which promote the accumulation of eosinophils and AAM. Deletion of ILC2s causes significant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associated with increased adipose ILC2 cytokine production and enhanced insulin sensitivity. Further, IL-33, a cytokine previously shown to promote cytokine production by ILC2s, leads to rapid ILC2-dependent increases in VAT eosinophils and AAMs. Thus, ILC2s are resident in VAT and promote eosinophils and AAM implicated in metabolic homeostasis, and this axis is enhanced during Th2-associated immune stimulation.

The environmentally widespread polysaccharide chitin is degraded and recycled by ubiquitous bacterial and fungal chitinases. Although vertebrates express active chitinases from evolutionarily conserved loci, their role in mammalian physiology is unclear. We show that distinct lung epithelial cells secrete acidic mammalian chitinase (AMCase), which is required for airway chitinase activity. AMCase-deficient mice exhibit premature morbidity and mortality, concomitant with accumulation of environmentally derived chitin polymers in the airways and expression of pro-fibrotic cytokines. Over time, these mice develop spontaneous pulmonary fibrosis, which is ameliorated by restoration of lung chitinase activity by genetic or therapeutic approaches. AMCase-deficient epithelial cells express fibrosis-associated gene sets linked with cell stress pathways. Mice with lung fibrosis due to telomere dysfunction and humans with interstitial lung disease also accumulate excess chitin polymers in their airways. These data suggest that altered chitin clearance could exacerbate fibrogenic pathways in the setting of lung diseases characterized by epithelial cell dysfunction.

The perinatal period is a critical window for distribution of innate tissue-resident immune cells within developing organs. Despite epidemiologic evidence implicating the early-life environment in the risk for allergy, temporally controlled lineage tracing of group 2 innate lymphoid cells (ILC2s) during this period remains unstudied. Using complementary fate-mapping approaches and reporters for ILC2 activation, we show that ILC2s appeared in multiple organs during late gestation like tissue macrophages, but, unlike the latter, a majority of peripheral ILC2 pools were generated de novo during the postnatal window. This period was accompanied by systemic ILC2 priming and acquisition of tissue-specific transcriptomes. Although perinatal ILC2s were variably replaced across tissues with age, the dramatic increases in tissue ILC2s following helminth infection were mediated through local expansion independent of de novo generation by bone marrow hematopoiesis. We provide comprehensive temporally controlled fate mapping of an innate lymphocyte subset with notable nuances as compared to tissue macrophage ontogeny.

Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells prominent at barrier sites. Although precursors are found in blood, mature ILC2s can enter the circulation after small intestinal perturbation by migratory helminths and move to distant tissues to influence the local reparative response. Using fate-mapping and methods to bypass the lung or intestinal phases of Nippostrongylus brasiliensis infection, we show that blood ILC2s comprise heterogeneous populations derived from distinct tissues that are dependent on alarmins matched to the receptor profile of the specific tissue ILC2s. Activation of local ILC2s by tissue-specific alarmins induced their proliferation, lymph node migration, and blood dissemination, thus systemically distributing type 2 cytokines. These studies uncover a possible mechanism by which local innate responses transition to systemic type 2 responses by extrusion of activated sentinel ILC2s from tissue into the circulation.

Type 2 T helper (TH2) cells are protective against parasitic worm infections but also aggravate allergic inflammation. Although the role of dendritic cells (DCs) in TH2 cell differentiation is well established, the underlying mechanisms are largely unknown. Here, we show that DC induction of TH2 cells depends on membrane-associated RING-CH-1 (MARCH1) ubiquitin ligase. The pro-TH2 effect of MARCH1 relied on lymph node (LN)–resident DCs, which triggered T cell receptor (TCR) signaling and induced GATA-3 expression from naïve CD4+ T cells independent of tissue-driven migratory DCs. Mice with mutations in the ubiquitin acceptor sites of MHCII and CD86, the two substrates of MARCH1, failed to develop TH2 cells. These findings suggest that TH2 cell development depends on ubiquitin-mediated clearance of antigen-presenting and costimulatory molecules by LN-resident DCs and consequent control of TCR signaling.

The small intestinal tuft cell-ILC2 circuit mediates epithelial responses to intestinal helminths and protists by tuft cell chemosensory-like sensing and IL-25-mediated activation of lamina propria ILC2s. Small intestine ILC2s constitutively express the IL-25 receptor, which is negatively regulated by A20 (Tnfaip3). A20 deficiency in ILC2s spontaneously triggers the circuit and, unexpectedly, promotes adaptive small-intestinal lengthening and remodeling. Circuit activation occurs upon weaning and is enabled by dietary polysaccharides that render mice permissive for Tritrichomonas colonization, resulting in luminal accumulation of acetate and succinate, metabolites of the protist hydrogenosome. Tuft cells express GPR91, the succinate receptor, and dietary succinate, but not acetate, activates ILC2s via a tuft-, TRPM5-, and IL-25-dependent pathway. Also induced by parasitic helminths, circuit activation and small intestinal remodeling impairs infestation by new helminths, consistent with the phenomenon of concomitant immunity. We describe a metabolic sensing circuit that may have evolved to facilitate mutualistic responses to luminal pathosymbionts.

Innate lymphoid cells (ILCs) are tissue-resident effectors poised to activate rapidly in response to local signals such as cytokines. To preserve homeostasis, ILCs must employ multiple pathways, including tonic suppressive mechanisms, to regulate their primed state and prevent inappropriate activation and immunopathology. Such mechanisms remain incompletely characterized. Here we show that cytokine-inducible SH2-containing protein (CISH), a suppressor of cytokine signaling (SOCS) family member, is highly and constitutively expressed in type 2 innate lymphoid cells (ILC2s). Mice that lack CISH either globally or conditionally in ILC2s show increased ILC2 expansion and activation, in association with reduced expression of genes inhibiting cell-cycle progression. Augmented proliferation and activation of CISH-deficient ILC2s increases basal and inflammation-induced numbers of intestinal tuft cells and accelerates clearance of the model helminth, Nippostrongylus brasiliensis, but compromises innate control of Salmonella typhimurium. Thus, CISH constrains ILC2 activity both tonically and after perturbation, and contributes to the regulation of immunity in mucosal tissue.

Programs defining tissue-resident macrophage identity depend on local environmental cues. For alveolar macrophages (AMs), these signals are provided by immune and nonimmune cells and include GM-CSF (CSF2). However, evidence to functionally link components of this intercellular cross talk remains scarce. We thus developed new transgenic mice to profile pulmonary GM-CSF expression, which we detected in both immune cells, including group 2 innate lymphoid cells and γδ T cells, as well as AT2s. AMs were unaffected by constitutive deletion of hematopoietic Csf2 and basophil depletion. Instead, AT2 lineage-specific constitutive and inducible Csf2 deletion revealed the nonredundant function of AT2-derived GM-CSF in instructing AM fate, establishing the postnatal AM compartment, and maintaining AMs in adult lungs. This AT2-AM relationship begins during embryogenesis, where nascent AT2s timely induce GM-CSF expression to support the proliferation and differentiation of fetal monocytes contemporaneously seeding the tissue, and persists into adulthood, when epithelial GM-CSF remains restricted to AT2s.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.