ILC2 Differentiation Research Articles

Microbiota regulates the TET1-mediated DNA hydroxymethylation program in innate lymphoid cell differentiation

Innate lymphoid cell precursors (ILCPs) develop into distinct subsets of innate lymphoid cells (ILCs) with specific functions. The epigenetic program underlying the differentiation of ILCPs into ILC subsets remains poorly understood. Here, we reveal the genome-wide distribution and dynamics of the DNA methylation and hydroxymethylation in ILC subsets and their respective precursors. Additionally, we find that the DNA hydroxymethyltransferase TET1 suppresses ILC1 but not ILC2 or ILC3 differentiation. TET1 deficiency promotes ILC1 differentiation by inhibiting TGF-β signaling. Throughout ILCP differentiation at postnatal stage, gut microbiota contributes to the downregulation of TET1 level. Microbiota decreases the level of cholic acid in the gut, impairs TET1 expression and suppresses DNA hydroxymethylation, ultimately resulting in an expansion of ILC1s. In adult mice, TET1 suppresses the hyperactivation of ILC1s to maintain intestinal homeostasis. Our findings provide insights into the microbiota-mediated epigenetic programming of ILCs, which links microbiota-DNA methylation crosstalk to ILC differentiation.

Nr4a1 marks a distinctive ILC2 activation subset in the mouse inflammatory lung

BackgroundGroup 2 innate lymphoid cells (ILC2s) are critical sources of type 2 cytokines and represent one of the major tissue-resident lymphoid cells in the mouse lung. However, the molecular mechanisms underlying ILC2 activation under challenges are not fully understood.ResultsHere, using single-cell transcriptomics, genetic reporters, and gene knockouts, we identify four ILC2 subsets, including two non-activation subsets and two activation subsets, in the mouse acute inflammatory lung. Of note, a distinct activation subset, marked by the transcription factor Nr4a1, paradoxically expresses both tissue-resident memory T cell (Trm), and effector/central memory T cell (Tem/Tcm) signature genes, as well as higher scores of proliferation, activation, and wound healing, all driven by its particular regulons. Furthermore, we demonstrate that the Nr4a1+ILC2s are restrained from activating by the programmed cell death protein-1 (PD-1), which negatively modulates their activation-related regulons. PD-1 deficiency places the non-activation ILC2s in a state that is prone to activation, resulting in Nr4a1+ILC2 differentiation through different activation trajectories. Loss of PD-1 also leads to the expansion of Nr4a1+ILC2s by the increase of their proliferation ability.ConclusionsThe findings show that activated ILC2s are a heterogenous population encompassing distinct subsets that have different propensities, and therefore provide an opportunity to explore PD-1's role in modulating the activity of ILC2s for disease prevention and therapy.

ILC2 regulates hyperoxia-induced lung injury via an enhanced Th17 cell response in the BPD mouse model

BackgroudRecent research has focused on the role of immune cells and immune responses in the pathogenesis of bronchopulmonary dysplasia (BPD), but the exact mechanisms have not yet been elucidated. Previously, the key roles of type 2 innate lymphoid cells (ILC2) in the lung immune network of BPD were explored. Here, we investigated the role Th17 cell response in hyperoxia-induced lung injury of BPD, as well as the relationship between ILC2 and Th17 cell response.MethodsA hyperoxia-induced BPD mouse model was constructed and the pathologic changes of lung tissues were evaluated by Hematoxylin-Eosin staining. Flow cytometry analysis was conducted to determine the levels of Th17 cell, ILC2 and IL-6+ILC2. The expression levels of IL-6, IL-17 A, IL-17 F, and IL-22 in the blood serum and lung tissues of BPD mice were measured by ELISA. To further confirm the relationship between ILC2 and Th17 cell differentiation, ILC2 depletion was performed in BPD mice. Furthermore, we used immunomagnetic beads to enrich ILC2 and then flow-sorted mouse lung CD45+Lin-CD90.2+Sca-1+ILC2. The sorted ILC2s were injected into BPD mice via tail vein. Following ILC2 adoptive transfusion, the changes of Th17 cell response and lung injury were detected in BPD mice.ResultsThe expression levels of Th17 cells and Th17 cell-related cytokines, including IL-17 A, IL-17 F, and IL-22, were significantly increased in BPD mice. Concurrently, there was a significant increase in the amount of ILC2 and IL-6+ILC2 during hyperoxia-induced lung injury, which was consistent with the trend for Th17 cell response. Compared to the control BPD group, ILC2 depletion was found to partially abolish the Th17 cell response and had protective effects against lung injury after hyperoxia. Furthermore, the adoptive transfer of ILC2 enhanced the Th17 cell response and aggravated lung injury in BPD mice.ConclusionsThis study found that ILC2 regulates hyperoxia-induced lung injury by targeting the Th17 cell response in BPD, which shows a novel strategy for BPD immunotherapy.

Identifying the expression and function of Blimp-1 in ILC2s in allergic asthma

Abstract Allergic asthma is driven by type 2 inflammation from Th2 cell and Type 2 innate lymphoid cells (ILC2s). We recently demonstrated a critical role for Blimp-1, a transcriptional repressor, in Th2 cells to promote allergic asthma. However, the expression and function of Blimp-1 in ILC2s remains undefined. Using genetically modified mouse strains that report Blimp-1 expression via YFP, we surveyed Blimp-1 expression in allergic asthma and other type 2 immune models across various tissues. We found that Blimp-1 is induced in ILC2s in the lung in response to the typical alarmins IL-33 and IL-25, as well as house dust mite (HDM). However, ILC2 activation did not upregulate Blimp-1 in the skin or gut in response to peanut allergen, suggesting a tissue-specific requirement for Blimp-1 in ILC2s. IL-33, as known to be a potent activator of ILC2s, was not required for Blimp-1 expression upon HDM stimulation. Interestingly, IL-10, a potent driver of Blimp-1 in Th2 cells, induced Blimp-1 expression in ILC2s, suggesting IL-10 but not IL-33 may be important for a Blimp-1+ ILC2 state. Intriguingly, Blimp-1 expression did not correlate with cytokine production, potentially representing a unique functional state of ILC2 cells that are high for Blimp-1. To evaluate Blimp-1 function, we deleted Blimp-1 in ILC populations using IL-7Ra Cre crossed to Blimp-1 floxed mice and generated mixed bone marrow chimeras with IL-7Ra Cre control donors. Blimp-1 knockout ILC2s in response to HDM exhibited an alteration in markers of ILC2 activation including ST2, KLRG1 and GATA3, suggesting Blimp-1 plays a role in promoting ILC2 differentiation. Collectively, our study has identified a lung-specific state of ILC2s expressing Blimp-1 that are found in a model of allergic asthma.

Strength of CAR signaling reveals bifurcation of T and ILC2 lineage differentiation from pluripotent stem cells

Abstract T cells and type 2 innate lymphoid cells (ILC2) are two closely related lymphoid lineages that share certain developmental and transcriptional programs, including a requirement for Notch and IL-7 signaling during differentiation. We recently developed an artificial thymic organoid (ATO) system that supports in vitro differentiation of human pluripotent stem cells (PSCs) to mature αβT cells. We show here that lentiviral introduction of a CD19-targeted chimeric antigen receptor (CAR) into PSCs surprisingly resulted in ILC2-biased lymphopoiesis from PSCs at the expense of T cell differentiation. PSC-derived ILC2s expressed the classical ILC2 markers CD25, CD200R, and CRTH2 as well GATA3, ID2, and TCF7, and responded to both cytokine stimuli and antigen-dependent CAR signaling by producing IL-5 and IL-13. To understand CAR-mediated lineage diversion from T to ILC2, we performed single cell RNAseq of PSC-derived hematopoietic progenitor cells before the phenotypic onset of either T or ILC2 differentiation. This revealed a gene signature of T cell receptor signaling, suggesting aberrant CAR activation at the earliest stages of lymphocyte development. We established that CAR signaling in ATOs was antigen-independent and thus likely driven by tonic signaling. We applied this finding to rationally modulate the T/ILC2 lineage decision by fine-tuning CAR signaling strength during lymphocyte development from PSCs, permitting restoration of conventional CAR-T cell differentiation or, conversely, directed differentiation of isogenic antigen-specific CAR-ILC2s. Taken together, our findings shed light on human ILC2 development and inform the applied differentiation of both ILC2s and conventional CAR-T cells from PSCs. Supported by Tobacco-Related Disease Research Program Predoctoral Award (SL), Broad Stem Cell Research Center UCLA Fellowship (SL, CSS), CIRM Bridges Program (CB), NIH T32 (ST), NIH K08CA235525 (CSS).

Dual JAK2/Aurora kinase A inhibition prevents human skin graft rejection by allo-inactivation and ILC2-mediated tissue repair

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100significantly reduces the frequency of skin-homing CLA+ donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells. We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.

TLR3-driven IFN-β antagonizes STAT5-activating cytokines and suppresses innate type 2 response in the lung

Group 2 innate lymphoid cells (ILC2s) are involved in type 2 immune responses in mucosal organs and are associated with various allergic diseases in humans. Studies are needed to understand the molecules and pathways that control ILC2s. The aims of this study were to develop a mouse model that limits the innate type 2 immune response in the lung and to investigate the immunologic mechanisms involved in regulation of lung ILC2s. Naive BALB/c mice were administered various Toll-like receptor agonists and exposed intranasally to the fungal allergen Alternaria alternata. The mechanisms were investigated using gene knockout mice as well as cultures of lung cells and isolated lung ILC2s. Polyinosinic-polycytidylic acid, or poly (I:C), effectively inhibited innate type 2 response to A alternata. Poly (I:C) promoted production of IFNα, -β, and -γ, and its inhibitory effects were dependent on the IFN-α/β receptor pathway. IFN-β was 100 times more potent than IFN-α at inhibiting type 2 cytokine production by lung ILC2s. Signal transducer and activator of transcription 5 (STAT5)-activating cytokines, including IL-2, IL-7, and thymic stromal lymphopoietin, but not IL-33, promoted survival and proliferation of lung ILC2s invitro, while IFN-β blocked these effects. Expression of the transcription factor GATA3, which is critical for differentiation and maintenance of ILC2s, was inhibited by IFN-β. IFN-β blocks the effects of STAT5-activating cytokines on lung ILC2s and inhibits their survival and effector functions. Administration of IFN-β may provide a new strategy to treat diseases involving ILC2s.

A Gata3 enhancer necessary for ILC2 development and function

The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.

POS0326 ROLE OF THE IL-25 / IL-17RB AXIS IN TH9 POLARIZATION IN PATIENTS WITH PROGRESSIVE SYSTEMIC SCLEROSIS

Background:Systemic sclerosis (SSc) is an inflammatory connective tissue disease leading to chronic and progressive fibrosis, typically affecting the skin and internal organs. The alteration of both innate and adaptive immune responses plays a pivotal role in SSc pathophysiology, although it has not yet been fully elucidated [1].Recent findings have demonstrated interleukin (IL)-9 overexpression and significant group 2 innate lymphoid cells (ILC2) expansion in patients with SSc. Th9-ILC2-mast cells axis seems to be involved in SSc tissue damage and in the induction of fibrosis [2]. Activation and production of IL-9 by Th9 cells are promoted by transforming growth factor (TGF)-β, thymic stromal lymphopoietin (TSLP), IL-25 and IL-33. Thus, the IL-25 / IL-17RB pathway would act as a key player in SSc.Objectives:The purpose of this study was to evaluate the role of the IL-25 / IL-17RB axis as a driver in Th9 polarization and ILC2 expansion and polarization in SSc patients.Methods:26 patients were enrolled in this study. Peripheral blood and skin biopsy specimens were obtained from SSc patients. PBMCs were isolated and incubated with and without recombinant (r)IL-25 for 24-48-72 hours and the frequencies of Th9 cells, Th17 cells and ILC2 were assessed by flow cytometry analysis. Moreover, the ex vivo expression of IL-17RB in ILC2 was also assessed. Immunofluorescence analysis was performed on biopsy skin samples to evaluate IL-17RB expression in ILC2.Results:In SSc samples, Th9 cells frequency progressively increased after stimulation with rIL-25, compared to healthy controls in which IL-9 frequency decreased over time regardless of rIL-25.Simultaneously, we evaluated the role of the IL-25 / IL-17RB axis in Th17 cells.In the SSc pool, the initially low rate of IL-17 increased at 72 hours after stimulation with rIL-25. In unstimulated SSc samples, the initially higher IL-17 rate decreased at 72 hours; conversely, it was consistently low in healthy controls, at both baseline and stimulated conditions.Our results confirmed the presence of IL-25-dependent clonal ILC2 expansion, suggesting a greater and progressive expansion over time in patients with SSc, compared to controls.Interestingly, increased IL-17RB expression was found in circulating ILC2 from SSc patients supporting the characterization of ILC2 inflammatory phenotype.Consistently, immunofluorescence on the skin of SSc patients showed a marked infiltrate of CD3-GATA3+ IL-17RB+ cells, confirming the presence of the activated inflammatory phenotype ILC2, absent in skin biopsies of healthy controls (Figure 1).Conclusion:These preliminary data suggest an active role of the IL-25/IL-17RB axis in SSc. It results in Th9 polarization and Th17 clonal expansion, inducing the production of IL-9 and, to a lesser extent, IL-17. Moreover, in addition to promoting Th9-mediated ILC2 differentiation, IL-25 directs the polarization of ILC2 towards the inflammatory phenotype.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 can arise in the embryonic thymus, from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 migrated to mucosal tissues with colonization of the intestinal lamina propria. RORα expression repressed T cell development, while promoting ILC2 in the thymus. From RNA-seq, ATAC-seq and ChIP-seq data we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 from common progenitors in the thymus. When Notch signaling is present, Bcl11b dampens Nfil3/Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the Nfil3/Id2 repression, allowing Id2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

The Inhibition of Group II Innate Lymphoid Cell Response by IL-27 in Allergic Rhinitis.

Objectives Interleukin-27 (IL-27) has been reported to inhibit type 2 T helper cell (Th2) response in allergic rhinitis (AR). However, its effects on group II innate lymphoid cells (ILC2) in AR are not fully understood. Methods Nineteen patients with AR and nineteen controls were enrolled in this study. The effects of IL-27 on ILC2 differentiation and function as well as the regulation of the IL-27 receptor (IL-27R) were analyzed by tritiated thymidine incorporation, enzyme-linked immunosorbent assay (ELISA), and real-time polymerase chain reaction (PCR), respectively. AR mice were used to confirm the role of IL-27 in vivo. Results The serum IL-27 protein expression in AR patients was significantly lower compared with controls. IL-27 decreased the ILC2 proliferation and type II cytokine secretion through the interaction with IL-27R. IL-27 also inhibited systemic and nasal ILC2 response of AR mice. Conclusion IL-27 inhibited the proliferation and function of ILC2 in AR, implying that IL-27 may be used as new treatment target in AR.

Imperfect storm: is interleukin-33 the Achilles heel of COVID-19?

The unique cytokine signature of COVID-19 might provide clues to disease mechanisms and possible future therapies. Here, we propose a pathogenic model in which the alarmin cytokine, interleukin (IL)-33, is a key player in driving all stages of COVID-19 disease (ie, asymptomatic, mild–moderate, severe–critical, and chronic–fibrotic). In susceptible individuals, IL-33 release by damaged lower respiratory cells might induce dysregulated GATA-binding factor 3-expressing regulatory T cells, thereby breaking immune tolerance and eliciting severe acute respiratory syndrome coronavirus 2-induced autoinflammatory lung disease. Such disease might be initially sustained by IL-33-differentiated type-2 innate lymphoid cells and locally expanded γδ T cells. In severe COVID-19 cases, the IL-33–ST2 axis might act to expand the number of pathogenic granulocyte–macrophage colony-stimulating factor-expressing T cells, dampen antiviral interferon responses, elicit hyperinflammation, and favour thromboses. In patients who survive severe COVID-19, IL-33 might drive pulmonary fibrosis by inducing myofibroblasts and epithelial–mesenchymal transition. We discuss the therapeutic implications of these hypothetical pathways, including use of therapies that target IL-33 (eg, anti-ST2), T helper 17-like γδ T cells, immune cell homing, and cytokine balance.

E proteins orchestrate dynamic transcriptional cascades implicated in the suppression of the differentiation of group 2 innate lymphoid cells

Group 2 innate lymphoid cells (ILC2s) represent a subset of newly discovered immune cells that are involved in immune reactions against microbial pathogens, host allergic reactions, as well as tissue repair. The basic helix-loop-helix transcription factors collectively called E proteins powerfully suppress the differentiation of ILC2s from bone marrow and thymic progenitors while promoting the development of B and T lymphocytes. How E proteins exert the suppression is not well understood. Here we investigated the underlying molecular mechanisms using inducible gain and loss of function approaches in ILC2s and their precursors, respectively. Cross-examination of RNA-seq and ATAC sequencing data obtained at different time points reveals a set of genes that are likely direct targets of E proteins. Consequently, a widespread down-regulation of chromatin accessibility occurs at a later time point, possibly due to the activation of transcriptional repressor genes such as Cbfa2t3 and Jdp2 The large number of genes repressed by gain of E protein function leads to the down-regulation of a transcriptional network important for ILC2 differentiation.

Inhibited interleukin 35 expression and interleukin 35–induced regulatory T cells promote type II innate lymphoid cell response in allergic rhinitis

BackgroundInterleukin (IL)-35 and IL-35–producing regulatory T cells (iTr35) have been reported to inhibit TH2 response in allergic rhinitis (AR). However, its effects on type II innate lymphoid cells (ILC2) are not well characterized. ObjectiveTo investigate the effect of IL-35 on ILC2 in AR. MethodsA total of 25 patients with AR and 20 controls were recruited. The expression and regulation of IL-35 receptor in ILC2 were analyzed by real-time polymerase chain reaction. The effect of IL-35 on ILC2 differentiation and cytokine production was analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, iTr35 were cocultured with ILC2 to explore the effect of iTr35 on ILC2. The AR mice models were also established to confirm the role of IL-35 in vivo. ResultsThe patients with AR had decreased IL-35 expression and iTr35 proportion and increased ILC2 and type II cytokines compared with the controls. Notably, IL-35 inhibited ILC2 differentiation and type II cytokine production by regulating IL-12Rβ2 and gp130. IL-35 promoted the inducible costimulatory molecule expression by iTr35 and the inducible costimulatory molecule ligand expression by ILC2. IL-35–treated mice with AR presented decreased frequency and function of nasal ILC2. ConclusionIL-35 inhibited ILC2 responses directly or through mutual contact between iTr35 and ILC2 in AR, suggesting that IL-35 may be used as a potential treatment target in AR.

LncRNA AK085865 depletion ameliorates asthmatic airway inflammation by modulating macrophage polarization

Accumulating evidence indicates that regulators of macrophages polarization may play a key role in the development of allergic asthma (AA). However, the exact role of long non-coding RNAs (lncRNAs) in regulating in macrophages polarization in the pathogenesis of dermatophagoides farinae protein 1(Der f1)-induced AA is not fully understood. The purpose of this study was to determine the function of lncRNA AK085865 in regulating macrophages in AA. Here we report that lncRNA AK085865 served as a critical regulator of macrophages polarization and reduced the pathological progress of asthmatic airway inflammation. In response to the challenge of Der f1, AK085865−/− mice displayed attenuated allergic airway inflammation, including decreased eosinophil in BALF and reduced production of IgE, which were associated with decreased mucous glands and goblet cell hyperplasia. In addition, Der f1-treated AK085865−/− mice show fewer M2 macrophages when compared with WT asthmatic mice. After adopting bone marrow-derived macrophages (BMDM, M0) from WT mice, Der f1-treated AK085865−/− mice also revealed a light inflammatory reactions. We further observed that the percentage of type II innate immune lymphoid cells (ILC2s) decreased in AK085865−/− asthmatic mice. Moreover, M2 macrophages helped promote the differentiation of ILC2s, probably through the exosomal pathway secreted by M2 macrophages. Taken together, these findings reveal that AK085865 depletion can ameliorate asthmatic airway inflammation by modulating macrophage polarization and M2 macrophages can promote the differentiation of innate lymphoid cells progenitor (ILCP) into ILC2s.

Leptin Regulated ILC2 Cell through the PI3K/AKT Pathway in Allergic Rhinitis.

Background Recent studies suggest that leptin is involved in Th2 response in allergic rhinitis (AR). However, the effect of leptin on type II innate lymphoid cells (ILC2s) in AR is not well characterized. Methods Twenty-six AR patients and 20 healthy controls were enrolled. Serum leptin levels were measured, and their correlation with ILC2 and type II cytokines were analyzed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. ILC2 differentiation and cytokine production stimulated by human recombinant leptin were analyzed by real-time polymerase chain reaction (PCR) and ELISA. AR mouse models were also established to verify the effect of leptin on ILC2 cell regulation. Results Our results showed that elevated serum leptin in AR patients was correlated with the percentage of ILC2 and the expression of type II cytokines. The recombinant leptin enhanced the expression of ILC2 cell transcription factors and type II cytokine through the PI3K/AKT pathway. The AR mice treated with leptin showed as stronger ILC2 inflammation and symptoms compared with control mice. Conclusions Our data provide evidence that upregulation of leptin promotes ILC2 responses in AR and this process was achieved through the PI3K/AKT pathway.

Epigenetic Plasticity of Type II Innate Lymphoid Cells in the Lower Gastrointestinal Tract

Though hematopoietic stem cell transplantation (HSCT) is the preferred treatment for a variety of blood malignancies, its use is limited by the development of acute graft-versus-host disease (aGvHD). Type II innate lymphoid cells (ILC2s) are immune cells that play an important role in maintaining mucosal homeostasis, and our lab has previously shown that ILC2s in the gastrointestinal tract (GI) are sensitive to conditioning therapy prior to HSCT. Strikingly, we have demonstrated that the infusion of activated donor ILC2s markedly reduces aGvHD-associated mortality. We therefore wanted to investigate the mechanism of the loss of protective ILC2s from the GI tract. We hypothesized that ILC2s fail to repopulate the gut after HSCT due to inflammatory environmental cues that convert ILC2 precursors to an alternate, ILC1- or ILC3-like fate. Thus, we evaluated the impact of cytokines associated with commitment on murine ILC2s by exposing them to cytokines that may promote differentiation to an ILC1 or ILC3 fate (IL-1b/IL-12/IFN-γ and TGF-b/IL-6/IL-23, respectively). We found ILC2 cells acquired the ability to secrete TNF and IL-17 after in vitro skewing (with these lineage-defining cytokines. To test the ability of these "ex-ILC2" cells to home to other tissues in vivo, GFP-ILC2s were infused into recipients at the time of transplantation. We tracked GFP-ILC2s to the liver and spleen, where they made IFN-g and IL-17 and expressed transcription factors associated with the ILC1 and ILC3 lineages (Figure 1). Next we assessed the ability of cytokines alter ILC2 fate via epigenetic reprogramming by using ChIP-sequencing to evaluate the presence of histone marks that may underlie cellular plasticity. We show that these changes are associated with alterations in epigenetic marks around pioneer, lineage-determining factors. We therefore chose to test a screen of compounds known to modulate a variety of epigenetic targets to ask if they can maintain or convert ILC2s to alternate fates and identified a number of compounds that target bromodomains, methyltransferases, and histonedeacetylases, respectively, that alter the viability and differentiation of ILC2s into an "ex-ILC2"-like phenotype. Preliminary work suggests that maintenance ofG9a expression is able to rescue the loss of ILC2s, which is being tested in vivo. Taken together, these data provide new insights into mechanisms by which innate lymphoid cell precursors are epigenetically regulated, providing novel approaches to treating aGvHD following HSCT. Figure 1 Disclosures Davis: Triangle Biotechnology: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Pattenden:Triangle Biotechnology, Inc.: Equity Ownership, Other: Inventor on intellectual property. Serody:Merck: Research Funding; GlaxoSmithKline: Research Funding.

IL-35 and IL-35-Induced Regulatory T Cells Inhibited Type II Innate Lymphoid Cells Response Through GATA3/RORα Pathway in Allergic Rhinitis

Background: IL-35 and IL-35 induced IL-35-producing regulatory T cells (iTr35) have been reported to inhibit Th2 response in allergic rhinitis (AR). However, the effect of IL-35 and iTr35 on type II innate lymphoid cells (ILC2) in AR is not well characterized. Objective: We aimed to explore the effect of IL-35 and iTr35 on type II innate lymphoid cells (ILC2) in AR. Methods: Twenty-five AR patients and 20 healthy controls were recruited. The proteint expression of IL-35, the proportion of ILC2, IL-4+ILC2, IL-5+ILC2, IL-13+ILC2 and IL-35+CD4+CD25+ cells were detected by enzyme-linked immunosorbnent assay (ELISA) and flow cytometry. The expression and regulation of IL-35 receptor in ILC2 were analyzed by real-time PCR. The effect of IL-35 on ILC2 differentiation and cytokine production were analyzed by real-time PCR and ELISA. The iTr35 were cocultured with ILC2 in Transwell or non-Transwell system to explore the regulation of the iTr35 on ILC2 differentiation and function using RT-PCR and ELISA. AR mice models were also established to confirm the role of IL-35 in the regulation of ILC2 in vivo. Results: AR patients had decreased IL-35 expression and iTr35 proportion and increased proportion of ILC2 and type II cytokines compared with controls (P<0.05). The recombinant IL-35 inhibited ILC2 differentiation and type II cytokine production through GATA3/RORα pathway by regulation of IL-12Rβ2 and gp130. IL-35 promoted the inducible costimulatory molecule (ICOS) expression by iTr35 and ICOS ligand (ICOSL) expression by ILC2. iTr35 inhibited the function of ILC2s through ICOS:ICOS ligand. IL-35 treated AR mice presented as inhibited ILC2 inflammation compared with control mice. Conclusions: IL-35 inhibited ILC2 responses directly or through mutual contaction between iTr35 and ILC2 in AR, suggesting that IL-35 may be used as potential treatment target in AR. Funding: This study was supported by grants from the National Natural Science Grant of China (No.81600785, No. 81700892, No.81970861), the Pearl River S&T Nova Program of Guangzhou (No.201710010085), Key Clinical Speciality of Guangzhou Women and Children’s Medical Center, Grant of Institute of Pediatrics of Guangzhou Women and Children’s Medical Center (YIP-2016-022, Pre-NSFC-2018-005). Declaration of Interest: The authors declare that they have no relevant conflicts of interest. Ethical Approval: This study protocols were approved by local ethical committee boards and written informed consent was obtained. All animal care and experimental protocols were approved by local ethics committee boards.

Generation of a novel GATA3 reporter and inducible deletion system to study GATA3hi Th2 and ILC2 cells in vivo

Abstract GATA3 is an important transcription factor for the development of CD4 T cells and innate lymphoid cells (ILCs); it is also essential for the differentiation and function of Th2 cells and ILC2s. To understand the physiological role of GATA3 in these cells particularly in vivo, we have generated a novel GATA3 reporter mouse strain by inserting the ZsGreen-T2A cassette in front of the translation initiation site of the Gata3 allele already carrying two loxp sites which allow inducible Gata3 deletion when it is crossed to the CreERT2 transgenic mouse strain. Our initial experiments with this mouse line showed that the reporter expression faithfully reflects the GATA3 protein expression in both in vitro differentiated Th subsets and in vivo isolated T cells and ILCs. Furthermore, the reporter expression was still observed even when the Gata3 exon 4 (encoding zinc fingers) was deleted by tamoxifen treatment. Thus, this novel mouse model will allow us to separate GATA3-deficient “Th2” cells and “ILC2s” during in vivo immune responses. We are now using this reporter system to explore the role of GATA3 in various cells types generated in vivo by comparing GATA3-sufficient and GATA3-deficient reporter positive cells.

BET Bromodomain Inhibitor iBET151 Impedes Human ILC2 Activation and Prevents Experimental Allergic Lung Inflammation.

Group 2 innate lymphoid cells (ILC2) increase in frequency in eczema and allergic asthma patients, and thus represent a new therapeutic target cell for type-2 immune-mediated disease. The bromodomain and extra-terminal (BET) protein family of epigenetic regulators are known to support the expression of cell cycle and pro-inflammatory genes during type-1 inflammation, but have not been evaluated in type-2 immune responses. We isolated human ILC2 and examined the capacity of the BET protein inhibitor, iBET151, to modulate human ILC2 activation following IL-33 stimulation. iBET151 profoundly blocked expression of genes critical for type-2 immunity, including type-2 cytokines, cell surface receptors and transcriptional regulators of ILC2 differentiation and activation. Furthermore, in vivo administration of iBET151 during experimental mouse models of allergic lung inflammation potently inhibited lung inflammation and airways resistance in response to cytokine or allergen exposure. Thus, iBET151 effectively prevents human ILC2 activation and dampens type-2 immune responses.